Halfway between Lady Looks Like a Dude and Dude Looks Like a Lady is the character who falls squarely in the middle of the male-female divide... except that no-one can decide which side the character actually belongs on. Lots of subterfuge always follows, as everyone follows him/her around to try and determine his/her sex once and for all. Expect a Gender-Blender Name, lots of Gender Neutral Writing and Pronoun Trouble. Usually, his/her gender will never be revealed, and no-one will be any the wiser. This type of character is also subject to The Un Reveal quite a few times.
The technical term for this kind of person is "androgyne", from the Greek for "man-woman." Used as an adjective, it's "androgynous".
There are various Real Life gender identities that match up with this trope. A number of people identify as both male and female (either both at once, or shifting from one to the other every so often), or a mix of the two, or as having no gender at all, or a third gender that doesn't fit into the male/female binary, or some combination of these things; these people are known as "genderqueer", an umbrella term for non-binary genders. Gender identity is distinct from one's physical sex: the majority of genderqueer people have typical male or female body parts, although some may be intersex in addition to having a non-binary gender identity (while some intersex people will identify as either male or female).
See also Viewer Gender Confusion, something which invoking this trope may cause to persist even if the character does get a Gender Reveal, and Crossdresser.
See also No Biological Sex, for characters who have no physical sex, and Hermaphrodite, for characters who have both. They may or may not look as androgynous as the ones whose gender is simply ambiguous. Can be one of the attractive things that leads to Everybody Wants the Hermaphrodite.
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Anime and Manga
In .hack; it's unknown if Helba's Player is a guy or a girl, or whether the player is a single person or a group using a collective identity.
Crona from Soul Eater. Japanese doesn't have to use gendered pronouns, and doesn't in this case. Any cases of "guy" or "daughter" in fan translations are so far just the translator's guesses. The author has stated that he neither knows nor cares what Crona's true gender is. The official English dub refers to Crona using male pronouns, but only because the writers (who are just as clueless) didn't want to resort using neutral pronouns 'it/they' because it would be insulting to Crona as a character.note Which happens to be Proper, since English is "supposed" to use male pronouns when gender isn't known. It also has Medusa refer to Crona as "my child" or "it" to avoid any gender orientation, with the later doubling as a Woolseyism since it reflects how she treats Crona. Likewise, all the other characters far more often say Crona's name instead of using he, she, him, or her. There is also a strong possibility that Crona is neither or both. Oddly, in-series Crona's androgyny is never commented upon except when Patty asks if Crona is a boy or girl when Kid fought Crona on the Nidhogg.
Yubel◊ in Yu-Gi-Oh! GX. No body except a demonic arm, uses 'boku' and initially primarily a female voice. Then, Yubel starts shifting from a female to a male voice and back, and also mixes that in with the voice of the host's. Characters also refer to him/her as, well, him and her. The answer, of course, is that Yubel is an intersex human/spirit fusion... living being. And we're not counting their past self...
The weirdest part is, Yubel was once human, was a friend of a young prince who would become Judai in a future life, and volunteered to become the demonic creature to act as his spirit guide. But even then, whether Yubel was a boy or a girl while human, despite being seen in a flashback, is hard to tell.
Kino, the main character from Kino's Journey, is pretty darn ambiguous. Kino's gender female is indirectly revealed in one episode, and in one of the movies Kino has a monologue about whether to use male or female pronouns (eventually deciding on "boku").
The Pokémon anime is one of the most notable examples, as most of the mons belonging to characters in the anime do not have their gender confirmed. Further complicating the issue (or the Isshu) are the ones that only have confirmed genders in one language. After that, viewers have to deal with whether to use male or female pronouns for the officially non-gendered mons (Ditto, Shedinja, a couple Steel-types, and most legendaries) as even the dubs have used genders in some cases.
Thankfully averted in the games, as every Pokémon has a gender listed in-game, except for those which can't have genders, starting in the Generation II games. Some of the anime examples can even be frustrating, as most Pokémon in the movies are telepathic. Even if the voice is clearly male or female, the characters will insist on saying "it". The only logical example of this is the eleventh movie's Shaymin, who transitions from a female voice to a male voice.
Ash's Pikachu. Arguments often occurred over his (now-confirmed) gender. Even the makers of the show didn't know what gender he was, until they decided to throw a Togepi with Attract at him and Meowth. Attract only works on Pokémon of the opposite gender; Meowth was confirmed male LONG ago, therefore the Togepi is female, therefore Pikachu is male (Attract affected him).
Attract quickly became the go-to move to confirm a Pokémon's gender in the anime. Emolga abused the hell out of it before Iris caught her (and many times afterward too), and Snivy had the move used against her by another female Snivy.
The Staryu and Starmie from the anime confuse the issue even further. Despite one evolving from the other, the Staryu has a male-sounding voice while the Starmie's voice sounds female. According to the Pokédex in Japanese version, Staryu is classified as a Hermaphrodite.
Belial from Angel Sanctuary. All the angels are supposed to pick a specific sex and take drugs to change their body to match. Belial quits partway through the process and ends up androgynous.
Possibly the most baffling example occurs in the dubbed movie Serendipity the Pink Dragon, in which the bird Peela-Peela is referred to as male in the first half of the movie and female in the second half. The voice actor seems to be female, though.
Hellsing's Heinkel Wolfe and Alucard's 'Girlycard' form in The Dawn.
Edward in Cowboy Bebop is introduced like this. Male name, lanky but with prepubescent features, typically dresses in a baggy white shirt and biker shorts (when in a dress, the effect is... sort of like drag). Regardless of attire, characters are often visibly confused, as Ed's behavior and voice reveal only childlike androgyny. In Ed's introductory episode, the ambiguity is resolved in the very last line, as we cut to an external shot of the ship while Faye wrestles with her:
Mobile Suit Gundam 00 has the Innovators AKA Innovades, a group of Artificial Humans, most of which are either sexless or genderless according to the people who made the show (excepting Anew, who's completely female).
An early episode in the second season has fun with this by having Tieria Erde, an Innovade and one of the main characters, go undercover at a fancy dress party in drag. Making it even funnier is his voice, which sounds just like a woman's, but is provided by...Hiroshi Kamiya, the actor who plays Tieria normally.
The 7-Tailed Beetle's host in Naruto. When the host comes back to life, they're confirmed as being a girl.
Mammon in Katekyo Hitman Reborn! of the Varia is stated to be a man but his future self looks suspiciously feminine. Also Daisy of Byakuran's Six Funeral Wreaths. Turns out to be a guy but for a while he was right in the middle.
Prunus Girl: Aikawa says he's a guy on a regular basis and still ends up ambiguous—he's really good at playing a girl, periodically hinting at Recursive Crossdressing and inviting Maki to personally confirm his gender.
Mr. 2 Bon Kurei from One Piece apparently thinks that he falls under this category, although he isn't very feminine looking (aside from the makeup and tutu). Emporio Ivankov and his Newkama army come a little closer by being able to change genders whenever he feels like it.
Haruta is a more straight case. Going with the way One Piece's non-Gonk female characters are usually drawn (ridiculous hourglass bodies and big irises), he/she is probably male, but One Piece does make exceptions to those two traits from time to time. And even if Haruta's a male, then he is a quite effeminate-looking one, which isn't typical of One Piece guys either. In the anime, he/she has a female Seiyuu, though that is, of course, not canon (he/she has no spoken lines in the manga).
Kurapika of Hunter × Hunter was this for a while until he was confirmed to be male, and there are many people who still aren't convinced. His tribal wardrobe and habit of dressing as females for undercover missions don't help things.
Nefelpitou as well, since unlike Kurapika nothing has been confirmed.
Alluka, who is referred to as female by Killua and male by Illumi to add to the confusion. This has made fans conclude that Alluka is male-to-female transsexual.
Toto of Deadman Wonderland does this on purpose, although he occasionally slips up and uses gendered language.
Played with: Being a fox spirit, Laon's gender changes to suit (and seduce) whoever is closest to him/her at the time.
Wandering Son has this with a few characters due to the manga's relation to transsexuality. Makoto is typically seen as a Camp GayWholesome Crossdresser rather then a Transsexual, though there's room for debate. Takatsuki had some mixed reactions with people due to her shyness, but it's been confirmed she's a trans boy.
In Black Lagoon it's impossible to tell which one of Hansel and Gretel is a boy and which is the girl as they change appearances for the heck of it. It is also speculated that they were both the same gender, which one is anyone's guess.
In 07-Ghost, Kuroyuri's gender has yet to be revealed. Even the other characters don't know, Kuroyuri threatens them with death if they tell.
In Family Compo Shion is automatically thought of as a woman for the early chapters. Then Shion announces the intent to go to university as a man. Shion's cousin then searches the family photo albums and realises Shion has been switching genders at every school attended. Attempts to see proof of Shion's sex has so far failed. Many fans presume Shion's sex is female (though Shion's final decision on gender is unknown).
The King in Arisa is this; the official translation uses the pronoun 'he' but notes that the Japanese pronoun is gender neutral, meaning the King can be female.
Arimaru from Gamaran has a masculine sounding name, but is mostly referred to with feminine pronouns (though others use masculine ones). However, unlike most of the examples on this page, this is not due to the fact that s/he's a gorgeous androgynous being but rather a grotesque, huge brute with dark skin and claw-like nails, and a really freaky face.
Drives the plot in Ouran High School Host Club, in which the Bifauxnen main character is mistaken for a boy by the rest of the cast at first, spends the entire series crossdressing and pretending to be one, and outright declares that they really doesn't care what gender people think of them as.
The titular character Maria Rose of Bara no Maria. A running gag in the series is whether Maria is a pretty guy or a pretty girl. It doesn't help Maria displays both typical boyish and girlish reactions and attitudes.
Inukashi, or Dogkeeper, from No. 6. His/her gender hasn't been confirmed. Though s/he uses one of the masculine Japanese Pronouns "Ore" to refer to him/herself, there are scenes which present him/her in a feminine light, most notably the one in which the child that Sion sends to him/her calls him/her "Mama" as s/he holds him.
Kuromaru in UQ Holder appeared to be this for some time, claiming to be male but with several hints of being female. Turns out he's from a species that has No Biological Sex until age sixteen, at which time they choose one and gain a form to match, and he anticipates picking male... but his form is still completely androgynous for now.
Frosch from Fairy Tail. All of the previous cats have clearly been male or female, as well as matched the gender of their dragon slayer companion. Frosch is given a fair few female indicators while paired with a male dragon slayer, and has an androgynous baby voice in the anime. When a question as to Frosch's gender came up on the Q&A section in the back of a volume, the people answering questions couldn't agree on what gender they thought Frosch was. Hiro Mashima has also told curious fans who contacted him over twitter that Frosch's gender is a secret.
Krazy Kat, per Word of God; Herriman described Krazy as "androgynous, but willing to be either". In-strip, Krazy did get called "him" often, but the mannerisms were either way. However, Krazy was female in the 1962-63 animated series.
One The Far Side strip showed a group of jellyfish in the ocean, with two outhouses, each with identical pictures of jellyfish on them. The caption below it reads "Only they know the difference".
Indigo of Sovereign Seven was said to be able to swap between male and female at will.
Desire of the Endless in The Sandman has no fixed sex, and is androgynous, fluctuates between sexes, or both. Being the anthropomorphic personification of desire, to the reader (and to presumably most people who perceive it) Desire usually shows a mix of traditional gendered traits, such as a suit and tie with feminine facial features and lipstick.
Played with in Tale of Solaron: being a Snake Person, Solaron's gender is hard for humans to tell, leading one of the party to convince the elf in the party that Solaron is female for a joke.
Brox in With Strings Attached. Brox seems like a five-year-old child who dresses to create an impression of indeterminate gender. Turns out the body is male and the soul is female. Which explains why the Baravadans use the genderless pronoun “sar” to refer to everyone.
The story does sort of hint that s/he is female, mentioning that s/he cannot take Wolfie's advice on principle because he is "a straight male."
S/he does also lead a horde of fangirls, with no definite males among their ranks.
The Guidestuck character Cal Strider. All that we know about his/her gender is that he/she got extremely angry at being called "Mr. Strider".
The Passion of the Christsubverted the traditional, unambiguously male image of Satan by having the character played by a woman with her head shaved and her voice altered in post-production to make it sound more masculine. Thomas Aquinas would approve: Satan, as a pure spirit, has no gender.
The archangel Gabriel in Constantine dresses as a man, but is played by Tilda Swinton, made to look as androgynous as possible.
Sadako Yamamura of Ringu is intersex; though she appears feminine, her genetic makeup is stated differently several times throughout the films, novels and manga.
In Rat Race, there's a barkeeper who confuses the cast and audience.
Man: Hey, miss!
(Barkeeper turns around; thin lips, hard face, doesn't look pleased, maybe for the "miss", one could guess.)
Man: Sorry, I thought you were a woman.
Barkeeper: I am a woman.
The protagonists of the "queer buddy movie" By Hook Or By Crook both have ambiguous gender identity: Bifauxnen, trans guys, or somewhere in between?
Briefly parodied in a scene of Mars Attacks!, with the person at the press conference who asks if the Martians have two genders, like humans.
Lord of the Rings: Gimli says, "Dwarf women are so alike in voice and appearance that they're often mistaken for dwarf men. This in turn has given rise to the belief that there are no dwarf women and that dwarves simply spring out of holes in the ground, which is of course ridiculous." (Aragorn adds to this, "It's the beards.")
The Ghost of Christmas Past from The Muppet Christmas Carol appears as a childlike creature whose gender is never stated. The spirit was a male in the original novel but voiced by a girl in the film.
In Star Wars cosmology, the Hutts are actually genderless (being hermaphrodites) but most are referred to by either "he" or "she" depending on the Hutt's own personal preference. (Referring to one as "it" is considered rude, and given what they tend to do to people who offend - or even annoy - them, not wise.)
Yuu Valentine from Project NRI. In-text, the pronoun used for Yuu is 'they'.
A... man? A woman? Noriko couldn't tell, but with their chiseled features and gentle demeanor, they were certainly very beautiful.
The Person of Indeterminate Gender from A Series of Unfortunate Events, who is called "he or she" or "it" even by other members of Count Olaf's troupe. The closest we get to a name is "The Big One".
Pwt in Muse Magazine. The subject is one of constant argument on the Muse Mail page. There was one cartoon in which the crow sings a limerick about how pleased he is Pwt, having a cold, can't keep up with him to chase him. He repeatedly refers to Pwt with male pronouns. Perhaps he just felt the need to use pronouns and flipped a coin or something, but it was once cited in a letter as proof Pwt is male.
Harry Potter's Blaise Zabini got a single mention in book one, which sparked years of furious debate over his or her gender. Fan Fiction summaries often stated Boy!Blaise or Girl!Blaise, each with their own set of conventions and fanon personalities. (Boy!Blaise was often suave and Italian, Girl!Blaise often red-haired.) The Dutch translator, who changed all the names, even thought Blaise was a girl and called "her" Bella in book one. In book six this was corrected and Blaise was called Benno instead.
Robin Hobb's Realm of the Elderlings sequence consists of three trilogies. In the Farseer trilogy we meet the Fool, who the narrator Fitz believes is male. The Liveship Traders trilogy has a totally different dramatis personae, including the female Amber. It's never stated that these two are the same person, but more and more clues are dropped as the trilogy progresses, and because Amber advised Althea how to disguise herself as a boy we start to think he/she is really female and was disguised as male in the earlier books. But in the Tawny Man trilogy, where we and Fitz are told outright that both characters are the same person, there's a section where Fitz inhabits the Fool's body and can presumably tell what sex he/she is, but never tells us! Fitz implies that the fool's kind are so different from humans that neither gender is appropriate — but then there's the Pale Woman to account for.
The Angel Islington of Neverwhere is so androgynously beautiful that it has no obvious gender and is referred to as "it." A few characters do call it a "he" at some points, but always go back to calling Islington an "it," implying that they only do so because they're not used to talking about a genderless being. The narrative persists in using the dehumanizing "it", which suggests a certain ambiguity about its nature.
Douglas Hofstadter's Metamagical Themas features a dialogue between people of intentionally unspecified gender, named Chris, Pat, and Sandy (interestingly, in the context of a discussion of the life of Alan Turing). When translated, the translators used epicene names in their own language, such as Dominique in French.
Dwarfs look very male. The twist is, they all look very male, and are mostly unconcerned with gender. There's a handful of known females (Gloria Thogsdaughter, Cheri Littlebottom, Lars Skulldrinker, Dee, and possibly Rhys Rhysson) and one known male (Cassanunda). Beyond that, any dwarf could be either. Basically, while the sex of a dwarf can be male or female, the actual gender of most is male.
The idea is played with in Feet of Clay, in which he introduces a dwarf keen on asserting her femininity (Cheri, above) with techniques such as makeup. However, she refuses to shave her beard because, though she is proud to be a female, she felt that doing so would be denying that she was a dwarf.
Inverted in the case of the golem that looks over Moist von Lipwig in Going Postal and Making Money. An employee who considers herself a Moral Guardian insists that only a female can clean the women's restroom, so Moist stuck a dress on one and called it "Gladys" to keep her quiet. It took its assigned sex to heart.
Also in Discworld, no-one knows what sex Great A'Tuin the Star Turtle is, although a lot of people are interested in finding out. It's obviously heading somewhere, what if it's migrating to a mating ground? Suppose it met another star turtle, would they fight or mate? If they mate, who's going to be on top?
In Bridge to Terabithia, Jess isn't sure whether Leslie's a boy or a girl when they first meet, and gets frustrated that the name could go either way as well, but guesses (correctly) that she's female. The book includes a picture of her, showing her looking very androgynous, with a boyish haircut.
In Raptor, Thorn (the main character) is intersex. "He" lives most of his life as a male but lived in a convent for a year and can easily pass as attractive members of both genders (voice midway, taller than some men but shorter than some women, etc). Early in the book, he deliberately dresses in ambiguous clothing, confusing his hunting companion who simply can't figure out his gender (and won't ask, since he figures Thorn is deliberately hiding it). He meets another one. They have lots of sex. Then the second one turns out to be a real bitch.
Taken to the extreme in Iron Council, where one of the characters, devotee of a god of secrets, doesn't even know his/her own gender. Followers of this deity forfeit knowledge about themselves to honor their patron, and this particular priest lost knowledge of what sex he/she happened to be. Self-examination can't clarify matters, as the character is blind to his/her own body features.
The gender of Hilary Tamar, the protagonist of a series of mystery stories by Sarah Caudwell, is never revealed.
In The Princess Diaries, there's a character called Perin in Mia's French class. Mia and her friends can't figure out whether Perin is male or female until their French teacher calls Perin a boy, leading poor Perin to have to point out in front of her entire class that she's actually a girl.
Nyumba in Someone Else's War. Actually, her gender is never officially stated, except that the narrator assumes she's a girl.
In The Android's Dream, Sam Wentworth is never referred to in gendered language. This was apparently unintentional on the author's part at first; later in writing, he noticed and decided to run with it. Word of God is that he himself doesn't know Sam's gender, or whether Sam has one.
H.P. Lovecraft, who took a great deal of inspiration from Poe, wrote several short stories, among them The Nameless City, The Festival, The Hound, Ex Oblivione, Cool Air, The Music of Erich Zann, and What the Moon Brings, all of which never explicitly state the gender of the protagonist.
Soulcatcher from the Black Company is this in the first book. 'Catcher is a centuries old sorcerer who always goes around in a leather outfit and mask which completely conceals their features, and their voice constantly shifts between numerous different ones, implied to be the voices of 'Catcher's victims. 'Catcher is usually referred to as "he" and considered male by the narration, but he's very slim and androgynous, and if you look at him closely enough you can see very slight curves that might be female hips and bust, or might just be a trick of the light. Soulcatcher is a woman, as revealed at the end of the book, and this is simply treated as a given in later volumes.
Live Action TV
One episode of Jonathan Creek has a police officer of unknown gender. Creek and Maddy spend the episode following him/her around to find out his/her name, to see which toilets he/she uses, etc. By the end of the episode both Creek and Maddy have reversed their opinions of the officer's gender, leaving them (and the audience) none the wiser.
the character, Sgt Richie, was played by a man. With really bad hair.
The Saturday Night Live character Pat appeared in multiple sketches devoted to this trope, ultimately scoring a feature film, It's Pat, where he/she meets another ambiguous character, Chris. Fans often point out one of Pat's last sketches, in which Pat tilts her head back slightly when kissed, as evidence that the character is a woman. This was unintentional by the actress, Julia Sweeney, but she decided that Pat was officially female for this reason.
Dr. Haru Tanaka on Bones has a deep voice and an unusual style of dress. No one uses any gendered pronouns without dispute to refer to Tanaka. This led Angela to ask:
Some fans have theorized that Angela lied because she had thought Dr. Tanaka was male and didn't want the others to know she was wrong. It doesn't help that Dr. Tanaka is actually played by a woman.
In the original Star Trek pilot episode "The Cage", the alien Talosians were played by actresses in heavy alien makeup, but their speech was dubbed over with male voices. The incongruity between their facial features and voices was intended to emphasize their alienness.
Next Gen: Riker dated an androgynous person once. The androgyne was being ostracised from her species' society for identifying as a woman instead of carefully maintaining gender ambiguous behaviour. It was an aesop about how transgender people should be accepted as whatever gender they identify as, even if that gender is ambiguous, but with "the masses" portrayed as being ambiguous rather than divided into two binary genders.
The actor, Jonathan Frakes, said that he'd have preferred it if the androgyne was played by an ambiguously gendered man, since having him kiss a female actor kind of nullified the point of the story.
Isabel on HawthoRNe. Aside from the name, it's hard to tell.
The sex of Hal and Lois' baby on Malcolm in the Middle was kept under wraps to the point that, when the writers couldn't just keep calling it "the baby", they revealed the name as Jamie.
A Mystery Science Theater 3000 episode featured a '50s "educational" short called Mr. B Natural, which had a woman in the title role. While "Mr. B" was (despite the name) pretty unambiguously female, Joel and the 'Bots had a lot of fun riffing over the character's presumed androgyny, and devoted a whole host-segment sketch to the question of exactly what (s)he was supposed to be.
The Other Wiki mentions that photo captions in C.G. Conn's magazine refer to the character as "he", adding to the confusion.
Id looks like a female (especially in the early art), dresses somewhat like a man, is regularly mistaken for a woman, loudly exclaims he's a man at every point, has strange costume folds that slightly hint of cleavage, and is quite careful to never expose the chest area.
In the original novels Id is unambiguously male. In the manahwa it has been made ambiguous probably for comedic effect.
The music video for "Lightning Crashes" by Live features an androgynous-looking angel (played by a woman) guiding human souls from death to rebirth.
David Bowie as well as other British glam rockers of the 70's.
Bowie also used this trope in his song "Rebel Rebel," where even the character's mother is "not sure if you're a boy or a girl."
Pink Floyd's David Gilmour came across as pretty androgynous at times. Observe.◊ (A friend of mine actually mistook him for a woman in that photograph; this is very notable in that she is a diehard Floyd fan who has a more-than-decent grasp on what each band member looks/looked like.)
Annie Lennox from the Eurythmics got some heat over looking a little more masculine than some would prefer.
Gorillaz bassist Murdoc has a pet raven named Cortez. The name is masculine and Murdoc refers to it with male pronouns, but he also mentions it laying eggs. Either he or the writers Fail Biology Forever. Then again, it's very hard to tell the gender of ravens, and it doesn't really matter except to other ravens.
Winner of 2014, Austria's Conchita Wurst is actually a man, but it's hard to guess from the first time seeing him.
Angels are often played as this, if not unambiguously female, Bishōnen or Pretty Boy, though in the original texts they are always referred to as male, possibly just for convenience sake because there is no clear reason why they would even have a gender anyway.
The Judeo-Christian God comes off as this to those who know enough Hebrew. Most if not all of "His" names and titles (such as Father and Lord) are male, and the religions are highly patriarchal, but many of the god's attributes such as "His" spirit, breath, presence or wings are described in feminine terms due to the Semitic languages' grammatical gender. It is not so noticeable in English due to its lack of gender outside of obviously male and female words, but even then God is described as a mother bear at one point. A bit less ambiguous in Christianity, due to many of its texts being in Greek (which doesn't have this ambiguity), the whole Father-Son dynamic - the truly ambiguous member of the Christian Trinity is the Holy Spirit, who is often depicted in a feminine form if given an kind of form at all.
Parodied in Dino Attack RPG. In a humorous shout-out to Team Fortress 2, there is a character named Pyro based off the pyro class. It was firmly established that the character was a man, but his mysterious nature led to some in-universe debates on his gender. When the mask finally came off, Pyro was apparently revealed to be a woman, but then it turned out that she was actually an imposter standing in for her father, the real agent Pyro.
In Unknown Armies, any avatar of the Mystic Hermaphrodite plays this role to the hilt. Particularly powerful avatars are even able to switch their physical gender daily, making the question unanswerable. The Freak, godwalker of the Mystic Hermaphrodite, is at the point where neither he nor she is the right word - and indeed no one seems to know which gender it used to be, if either.
The novel Godwalker reveals it used to be female.
The Chaos gods Slaanesh and Tzeentch in Warhammer and Warhammer 40,000. Though the other two major Chaos gods (Khorne and Nurgle) are identified as male, Slaanesh is referred to (among its many names) as both "She Who Thirsts" and "the Prince of Excess", while Tzeentch is so utterly incomprehensible it might as well be fifty genders at the same time.
Slaanesh is both and neither at the same time, Tzeentch, well he's normally a cloud of colors and is called a "he" to keep things simple.
Vampire: The Requiem features the Galloi, a Nosferatu bloodline that decides to trade in "the crawling creepies" for "beauty beyond compare." It doesn't quite work out as planned; they become beautiful and utterly androgynous, but they're so beautiful that they can't possibly be anything natural, and thus still unnerve people. On top of that, a lot of them worship Cybele, and prefer to do so the old-fashioned way.
A Mage: The Awakening sourcebook (Legacies: The Sublime) describes the Daksha, a Legacy of Mages who seek to ascend to a higher evolutionary stage. That higher form happens to be three-eyed and hermaphroditic, which apparently extends to personality and gender identity. To confuse matters further, they are able to shift to a biologically male or female form at will.
Also in Awakening, all but one of the ruling Exarchs are referred to alternately as male or female (the exception being the Father, who embodies the divine patriarch). Given that they sloughed off their physical forms to become Platonic ideals of power and subjugation, whatever gender they identified with as human is likely irrelevant.
Luna, Exalted's goddess of the Moon, is typically identified as female, but is also likely to show up as a hermaphrodite or a man. The Lunar Exalted's powers allow them to be female, male, or neutral as they see fit, and at least one canon Lunar no longer identifies with a particular gender.
In Dungeons & Dragons, there are the ormyrr, a non-humanoid race that look like huge worms with big, toad-like heads, and four arms. While they do have genders, it's impossible for members of other races to tell them apart. Most other races use neutral pronouns when referring to them (which doesn't offend them, apparently).
The Planeswalker Ashiok from Magic: The Gathering. This became a sort of contention when both the player's guide and the german translation reffered to it as "male", but the official stance is that its gender is ambiguous/nonexistent. Considering what exactly Ashiok is...
The play Sylvia, by A.R. Gurney, has a character named Leslie, a person of indeterminate gender, that asks the other characters to assign gender roles to him/her when they first meet.
Though this may not have been intentional, Mistoffelees from Cats could be seen as having an ambiguous gender, especially in some productions (including the DVD version) even though he is referred to as a "he". Mistoffelees tends to share the choreography of the female cats more than the toms, in several stage versions he isn't matched with anyone at the mating dance, and a line from his signature song implies that "he" has had kittens.
In the original T.S. Eliot poem, it's more obvious that Mistoffelees is actually a female cat. Not sure if this translates to the stage version, though.
Angel from Rent is physically male but dresses as a woman most of the time. Sometimes s/he refers to himself using male pronouns and sometimes with female ones. So do his friends and his love interest. It's left pretty open if s/he's a drag queen, trans, genderqueer, or none of the above.
In the Israeli otaku musical And Sushi For Free, the main protagonist is a child of about ten, allegedly the star of some anime, with the ambiguous name Daniel, who looks rather ambiguous, is addressed by people as either and doesn’t seem to mind. Some characters are confused about this with no reponse, which is somewhat of a running gag that’s quickly dropped, but later turns into a Chekhov's Gun. It is revealed to be the source of much Internet Backdraft on The Other Wiki...
All Beanie Babies made since 1996 come with a descriptive four-line poem on their tags. In most cases, it's obvious that a particular Beanie is canonically male or female: either the poem specifically uses "he" or "she", the name is clearly gender-specific, or the design is a giveaway. (For instance, there are several kangaroos; while only one explicitly uses "she" in the poem, all of the others except Austin are clearly female despite their non-gender-specific names, as they have joeys in their pouches.) However, there are just some cases where the name is gender-neutral and either the poem is in first-person, or it just manages to get to four lines without using a pronoun.
Every LEGO minifigure pre 90's could technicly qualify, espeacially when using the default "Yellow smily face". Though largely averted with the town themes that actually have molded hair and more varying faces.
The Lamias' "attract" attack works on him/her, so whatever s/he is, s/he's attracted to women. Shudder.
Zidane won't protect him/her when equipped with the Protect Girls ability. Of course, that just means he doesn't know himself.
Jamie, the player character's nemesis from Harvest Moon: Magical Melody. Jamie dresses in clothes that deliberately make him/her appear androgynous (he/she looks the same regardless of whether you choose to play as a boy or a girl, and is officially female if you are male and vice versa).
In an official art for him/her, the female Jamie looks more feminine. Not that you can tell ingame.
Often times your child has no official gender, mostly in earlier games, so you can chose its gender. It doesn't affect the game, and the games don't refer to any pronouns.
NiGHTS (from the game of the same name) is intersex and has "apparent gender" that's whatever would be appropriate for a particular viewer. It was given a British accent that sounds like a cross between a teenage girl and a little boy.
Arno from Summon Night: Swordcraft Story 2. Her/His/Its gender is never revealed and she/he/it calls her/him/itself a 'child of the wind'.
This isn't helped that in the English games, its voice is masculine, but in the original Japanese it's feminine.
The Japanese official site seems to suggest s/he is female by putting her/him with Dinah and Aera, while the confirmed male summon beasts are with Aera's male counterpart.
The Dragon Child Coral of Summon Night 4 is explicitly this as a direct result of Schrödinger's Gun. When the Dragon Child is first met, the protagonist is asked by another character what the Dragon Child's sex is. The response options are "male", "female", and "I don't know". Coral is the result of choosing the third option. Gameplay and Story Integration keeps this up as Coral ignores sex restrictions on equipment and can be used in both the Undead Ship Captain and Dryad collaborative summons, which requires 4 male characters and 4 female characters respectively.
Revan in Knights of the Old Republic is never referred to in the game as male or female because Revan is the player character, which can be male or female depending on the player's selection at the beginning. There is one line of dialogue, spoken by Juahni, that refers to Revan as female, but this was an error in the coding that missed an if/then note earlier in the game. Other characters, particularly Canderous, use male pronouns to refer to Revan — which is canon, as confirmed by The New Essential Chronology.
Frog: This is no ordinary woman! Meet Flea, the magician! Flea: What the...?! Hey, I'm a GUY! Robo: But its exterior is that of a female... Flea: Male... female... what's the difference? Power is beautiful, and I've got the power!
This is even more in context in the DS version:
Frog: She is a powerful magician. Do not lower your guard! Flea is not the mere woman she seems. Flea: Yes, yes... I'm a man, after all, right? *pout* Lucca: Say what? That's a guy!? Flea: Tee hee hee... Man or woman, it's all the same. Power is beauty, and I'm deliciously strong!
Chrono Cross has an even more interesting example. The computer program FATE is often referred to as the "Goddess of Fate", and in battle, it boasts feminine features. However, the target does say that it is male, and on top of that it had inhabited a male form for the entire game up until that point. Since it is actually a supercomputer, though, gender might be a moot point.
The Great Mizuti from Baten Kaitos. However, during a very important scene near the end of the game the truth is revealed: Mizuti's a girl.
Guillo from Origins might count since as an animated puppet, he/she doesn't really have a gender. Guillo's personality is actually a gestalt created from the lingering memories of a man and a woman. Guillo is both and neither at once.
Nergal's morphs in Fire Emblem: Blazing Sword are supposedly genderless, or maybe that's just Fanon. Gets applied to Limstella the most often, although Ephidel is probably just as ambiguous. Of course, if they really are genderless, that brings up a lot of questions regarding how Sonia could operate under the delusion of being human—or, for that matter, what type of relationship Sonia and Brendan had that he could not know that there was something seriously weird about his new wife. The latter can be Hand Waved with the fact that Sonia seemed to have Brendan under some sort of enchantment, anyway.
Sonia was also created for the specific purpose of being Brendan's wife and doesn't even know she's a morph. It's possible she has... non-standard equipment.
Considering how Sonia has some characteristics that Limstella seems to lack, and, as mentioned, Sonia was specially created, this lends quite a bit both to "most morphs are genderless" andhow both she and Brendan assumed she was human.
Applied to two of the playable classes in the first three Geneforge games, though the Agent is clearly female. Interestingly, while the first and second game have the same character model for the Shaper, the drawings accompanying the loading screens make "him" look more male in the first game and more female in the second. (The third solves the problem by showing the Agent in the drawings.)
Final Fantasy Tactics Advance also makes generic allies and enemies androgynous, with the exception of the Viera. Characters are randomly assigned male or female names, though the former seem to be more common.
Poison in Final Fight. The designers were worried about her, given that North American players might have objected to a character hitting a girl, so whether she was female, a male transvestite, or transsexual was debated for awhile. They finally settled on male-to-female transsexual in all continuities, the only difference being whether she's pre-op (Japan) or post (everywhere else that cares). (Of course, this ignores the fact that there were two female Mooks in the original game; Poison and Roxy were color swaps, and Roxy was definately female. A profile of the characters that claimed Poison was a man also addressed Roxy as a "she" who disliked Poison's cross-dressing.)
Zohar from Silhouette Mirage for PS1. He/She changes genders based on what powers it's using. Though it's a computer program so gender might be irrelevant.
The Legend of Zelda: Ocarina of Time has Sheik, who is Princess Zelda in disguise. However, fans are divided over whether or not Zelda made herself into a man, or simply disguised herself. The skin-tight body suit highlights certain physical features, such as pectoral muscles and... other areas. Fans of female Sheik claim this to be an armor of sorts beneath the bodysuit, despite this severely restricting her movements. The instruction manual in Super Smash Bros. Brawl officially states Sheik's gender to be female. Additionally, Sheik was given more feminine attributes, like longer hair and an absence of frontal muscles. However, as the Brawl Zelda characters are based on Twilight Princess, the jury is still out on Sheik from Ocarina of Time.
In Majora's Mask, Majora's gender is never stated and left ambiguous. Its final form is muscular like a male, yet the "eyes" of the mask resemble breasts, and it has a high-pitched voice. As a result, many fans dispute as to whether Majora is male or female, or whether it even has a gender at all.
Pyro of Team Fortress 2. At first, given that Pyro had the same voice actor as the clearly male Spy, and had generally male movements and mumbles, all indication were that Pyro was a guy. The confusion started in a line on the Pyro's profile that stated "...if he is even a man". This was apparently meant to be about the Pyro's evil personality (which turns out isn't entirely right), but several fans thought that meant the Pyro might be female. Later on the developers ran with it and an update added a flowery purse to the inventory (apparently as a lame reference to Pyro being a "flamer") leading to more confusion on the part of the user base. Since the full body-suit and mask prevented any real confirmation, Valve ran with the idea, editing the posts on the official blog, changing subtle references to the Pyro's gender, just to screw with people's minds. This ambiguity then carried into in-game with the pre-2011 menu interface, in which the class recommendation window has the seemingly innocent and consistent line "Why don't you give him a shot?" when recommending a class to the player... Except when the pyro appears, it changes randomly between "him" and "her". With the release of "Meet the Pyro", it has become clear Valve intends to keep the mystery of Pyro's gender just that. Word of God is "Not telling is funnier than anything we could say".
Although Word of God says that in 8-Bit Theater the reason White Mage's sprite isn't taken from FFI (unlike all the other initial class characters) is because FFI's White Mage sprite couldn't be female since he's obviously male post-class change. Ironically, in Updated Re-release versions of FFI, the White Wizard sprite is far more feminine than previously.
Hilariously enough, the sprite for White Mage was taken from FFIII, in which the White Mage is definitely male.
No one knows for sure what Seem's gender in Jak 3: Wastelander is. His/her body type is ambiguous, which is amplified by the many layers of clothing and armor s/he wears. S/he could be male, because s/he was originally scripted as such. Daxter refers to him/her as 'monk boy' and Seem never corrects him. The trophy for rescuing him/her at the temple is titled "dude in distress". In the Spanish, Italian, German and French versions of the game, Seem is stated to be male and is voiced by a male actor. A fan emailed Naughty Dog and the answer received was "Seem is male." And the official Jak 3 website gives his/her gender as male. But s/he could also be female, because s/he is voiced by Tara Strong. The URL showing the final concept art on Bob Rafei's website contains "Seem girl merged". Another fan emailed Naughty Dog as well and the answer they received was "Seem is a girl." And the creator commentary and official guide refer to him/her as female.
The Makeover Mage in RuneScape. The mage may appear, when you first see him/her, to be man or a woman. That's because the mage keeps switching between being a guy and being a girl, so it's impossible to tell his/her gender.
Ghost Trick: While he spends the entirety of the game presenting as and being referred to as male, there's some fandom debate as to Sissel's actual sex. This is partially due to his Gender-Blender Name and the fact that he was named after Yomiel's female fiancee. Since Sissel didn't even know he was a cat, it's possible he also forgot that he was female, since during her first ghost manifestation Lynne also thought she was male (and Cabanela-shaped).
This is a trait of the Lapine race in Pandora Saga; the race has no gender selection due to the fact that the males and females are almost impossible to tell apart. At one point the lapine host of the first trailer gets angry at the audience for not being able to tell. Naturally, the real answer is interrupted.
The first thing Subaru from Sakura Taisen V does in the game is respond to the protagonist wondering about Subaru's gender by saying that the "difference in organs" doesn't really matter, and "Subaru is Subaru". This ambiguity persists throughout the game; for every line that hints that Subaru is really female, there's another that suggests that Subaru's really male. In the English version, characters refer to Subaru as "she" (as does Subaru on one occasion, which seems a bit out of character, but it can't be easy to translate for an ambiguously-gendered character who's also a Third-Person Person); however, in the original Japanese Subaru is never referred to by a gendered pronoun at all. NIS America deals with this by referring to Subaru in a promotional ad as a "guygirl".
Tuta from Suikoden II isn't obviously a he, considering his sprite and character portrait. Indeed, some people didn't realize his actual gender until they saw him grown up in the sequel.
Anna Hottenmeyer of Mr. Driller is a girl that can get some people to believe she's a guy (even in her normal person get-up)
All the dogs in Dogs Life, besides Jake and Daisy. They all have Barbie Doll Anatomy and don't interact with Jake much, though you may hear a human refer to a few of them as being male or female (such as Lopez being a girl, the sheep-dog being female, and Snookie apparently being male).
World of Warcraft has had much fan debate over the gender of the bronze dragon Chronormu/Chromie. Due to the dragonflights' Theme Naming any name ending in -ormu is male while females are -ormi, but whenever he/she assumes a humanoid form it's always as a female gnome, and no other dragon has been shown shifting into a humanoid form of the opposite gender.
Word of God later confirmed her to be female, meaning that for her species, she had a Gender-Blender Name and goes with a somewhat more feminine-sounding name instead.
Vestera, a Vernal god/dess from Lusternia. Appropriately, his/her dominion is over illusions and dreams, so gender is less relevant anyway.
The protagonist of Pirouette is referred to as both a "daughter" and as a "husband" by various characters and has an androgynous appearance.
In The Elder Scrolls series, the Daedric princes only choose to appear with a gender, and some of them, such as Boethias, make full use of this trope.
Captain Viridian in VVVVVV is never explicitly assigned a gender. Even when giving (somewhat less-than-fulfilling) romantic advice to a trusted member of his/her crew, the issue of Viridian's gender is entirely unresolved in any capacity. The retro graphics do ultimately little to help clear anything up.
In Journey, the mysterious robed player character has no gender.
Megaman Juno, the Final Boss for the first Mega Man Legends game, courtesy of a feminine facial structure, long, pink tresses, and being named after a Roman goddess. While it's made known that Juno is male, western players were struck with Viewer Gender Confusion due to his voice actor's (also male) vaguely feminine voice (in the Japanese version, it's more obvious, being voiced by Akira Ishida and all).
Since Omochao is obviously a robot, it's probably neither.
A Running Gag in the Disgaea games is the gender of the Alraune class. They look like flat-chested females, but one character that tried to flirt with one had an Unsettling Gender-Reveal. Disgaea 3 and 4 also introduce them as male when you create one (though they don't mind if you prefer to think of them as girls). And they respond to gender specific in-game effects as female. Since they are plants, it's possible that they have no actual gender, and Rule of Funny applies.
You can play as a lady, a gentleman, or an individual of mysterious and indistinct gender in Fallen London. This option causes the NPCs to stutter in confusion ("sir- er, mad- er, yes") and just sort of give up when the dialogue calls for them to refer to the player character by gender. It also has its own gender portraits. It doesn't affect anything else, since the game has Purely Aesthetic Gender.
My dear sir, there are individuals roaming the streets of Fallen London at this very moment with the faces of squid! Squid! Do you ask them their gender? And yet you waste our time asking trifling and impertinent questions about mine? It is my own business, sir, and I bid you good day.
Additionally, the Rubbery Men are only referred to as 'men' by convention (the player character occasionally gets an impression that one of them is, in fact, female, but it's rather difficult to be sure), and the Masters of the Bazaar style themselves as businessmen with names like Mr Cups and Mr Iron but wear all-concealing black hooded cloaks which make it difficult to ascertain either their species or their gender. Both groups are commonly referred to as 'it'.
There's also the Muffled Intriguer, a minor character who you mainly interact with when trading Influence items. Their gender is ambiguous mainly because they wear layers upon layers of clothing, which obscures their identity as well as their sex.
The playable Old Axe Armor in Castlevania: Portrait of Ruin reuses Johnathan's voice clips for various actions, but enemies react to it as they would with a female character (e.g. the Zacchino enemy type offers it flowers rather than attempting to stab it). Official art exists◊ showing the character to be female, however.
Sabor is a female in her source film, but a male in Kingdom Hearts. It may be a journal error, but since she and her world have beenretconed out of existence, we will never really know.
Three Dream Eaters, Meow Wow, Meowjesty and Flowbermeow, are this trope. Their journal entries speak of whether they are male or female, or if they are cat or dog (or chicken in the latter's case). This is also true with most of the other Dream Eaters, but a few, such as the Kyroo triplets, are stated to be male.
Rche from beatmania IIDX 19 Lincle is heavily implied, but not outright stated, to be a crossdressing male. Rche bears a male symbol tattoo on the bellybutton and references to Rche's gender in supplemental material are mosaic-censored. Notably, one piece of side material describes Rche as a "[censored]の娘" (no ko, literally "daughter of"); this could be interpreted to mean Rche is the daughter of a character with a one-kanji name, or that Rche is an "男の娘" (otoko no ko, a slang term for a crossdressing male).
Shittyghost in Quest Fantasy. He's referred to as a boy in most of the games, but then in Love Plus Shoujo Edition she is inexplicably considered a girl. S/he is a ghost of a male character, though.
The P.E.K.K.A from Clash Of Clans is so heavily armored, no one knows if it's male or female. However, one of the hints at the game's loading screen implies that it might be female, though that might be a typo.
Robopon has Kamat, the Legend4. The game describes Kamat as male, but Nintendo Power says Kamat is female. His/her sprites, on the overworld and in-battle, do not help in any way whatsoever.
Oyashiro-sama from Higurashi: When They Cry. In the anime it's referred to as male. In the sound novels, as female since they're talking about Hanyuu, is female. Its statue is very gender neutral too, in a religious way.
Lion Ushiromiya has a deliberately ambiguous gender that is probably essential to the mystery itself. All the pronouns are deliberately written as ambiguous, and even the translations kept him/her perfectly ambiguous. Even the detective once wondered if he/she were a "fairly slender boy" or a "serious, no-nonsense girl". Lion refuses to answer him directly, and Bernkastel confirms that she hid Lion's gender on purpose.
By extension, Yasu, also known as Shannon/Kanon/Beatrice and Lion's Alternate Self, also has an ambiguous gender that is deliberately hidden from the readers. Since Yasu has two female personas (Shannon and Beatrice), one male persona (Kanon) and is stated to have appeared as a man in one game (the Man from 19 Years Ago), their true gender is essentially left to the audience. However, the manga heavily implies that Yasu was born male but Raised as the Opposite Gender.
And then there's Zepar and Furfur, twin demons who are stated to be of opposite genders, though it's never revealed which is which. They serve as a Greek Chorus in EP6 and EP7 and they represent Lion and Yasu's Ambiguous Genders, as well as Yasu's gender confusion as a whole.
In Super Dangan Ronpa 2, The Super High School Level Impostor's gender is unknown. They do spend the game disguised as Byakuya Togami, who is male, but they are somewhat androgynous in appearance.
Although he is unquestionably male in canon now that Star Fox Command has come out, the VG Cats comic "It's Pat" (named for the above example) gives this status to Slippy from Star Fox, with Aeris and Leo following him to the loo to try to identify his gender - only to be foiled when instead of going into the male or female toilets, he pees in the break room.
V's "mate" doesn't help, neither do his/her adopted children. V refers to his/her husband/wife (who looks just as ambiguous as V) as "my mate." His/her mate refers to him/her in the same way. And their kids (who are also ambiguous looking and adopted)? They call him/her "Other Parent" (They are only seen speaking a foreign language).
Note this actually complicates things even further, as not only do we not know V's gender, nor V's mate's gender, but we do not even know for certain whether they are each the same or of different genders.
For whatever it's worth, when Roy used the Belt of Gender Reversal to turn into a woman, Vaarsuvius didn't notice anything different about him.
The Director from Skins has feminine facial features but is flat-chested and wears male attire. Word of God states that the Director is a true shapeshifter so presumably he/she shifts gender along with form.
Styx. Although originally appearing by taking over several nominally female characters, Styx's later appearances, in physical, robot bodies, only get more and more ambiguous as Coga Suro progresses.
Fluffy from the No Fourth Wall segments of Dan and Mab's Furry Adventures . At one point, during 'Reader Mail', Amber receives a letter questioning what gender Fluffy is, causing Fluffy to complain that "that's too stupid, even for the readers", walking away complaining about their obliviousness... as soon as she/he/it's off-panel, Amber admits that she doesn't know either, despite having created it.
This is explainable by three facts. Firstly, "Cubi" is basically a template, and Mink's default species is "Mythos" — which is the In-Universe equivalent of Lovecraft LiteEldritch Abominations (to put it in perspective, one Mythos character basically looks like a giant blue cobra with a human face, three eyes, Non-Mammal Mammaries, three-fingered hands, and a body that looks like a cross between a snake and a centaur) — meaning that an aversion of Non-Mammal Mammaries may be applicable. Secondly, all Cubi have the innate ability to freely switch between genders at will, meaning Mink could be male, female or both whenever he wants. Finally, Mink is The Ditz and so has forgotten what he is.
Pippin from Stupidity in Magic, the bisexual Author Avatar's boyfriend/girlfriend. The other characters treat him as male, but admit at intervals that they're not really sure, and the happy couple's keeping mum.
Noah, from El Goonish Shive, whom we know as a boy only because it was said and in comparison to whom Tedd doesn't look "that androgynous" at all, despite this being an old Running Gag.
SPDA from Magicandphysics is ambiguous to the point the creators don't know.
Insecticomics has fun with this: the giant androgynous robots are whatever gender and gender role they feel like being at the time. Thrust even changed gender to female as a mocking response to Lady Jaye's complaints about gay robots—and has kept it that way ever since, though no other aspects of her personality have shifted.
Sydney Morgan from This Is Not Fiction is an anonymous romance novelist who the main character has fallen in love with. He's convinced that she's female but everyone else is not so sure.
Calmasis, a character in a book-within-a-webcomic (the comic being Homestuck) is said to be "androgynous" and referred to as s/he. Whether this means Calmasis merely chooses to conceal his/her gender, is a person of nonbinary gender identity, or is a non-human with No Biological Sex is as yet unknown.
Calliope and Caliborn, who share a body, both have specific genders they identify with (Calliope being female and Caliborn being male), but the body they share is very androgynous-looking, having huge eyelashes, no hair and being flat chested. However, you can tell them apart by the colour of their cheek swirls. Adult cherubs appear externally male and female, though in the example we're given, if the female wins their mating duel she lays the egg inside the male. Presumably, if she lost, the male would fertilize her.
Kanryl of Ears for Elves looks reasonably androgynous, though it seems many readers refer to "him" as male, and there is no word about what "his" gender actually is. Archmage has stated he's keeping a tally of how many people consider "him" male or female.
Riley from Sire is invisible and hides his/her gender for the lulz.
P. Monkey, the Companion Cube character from lonelygirl15, has been variously described as a "she", a "he" and an "it", a practice which is finally lampshaded in "I Miss Her" - Bree isn't sure whether to call P. Monkey a boy or a girl.
Masquerade, a shapeshifting supervillain from the Global Guardians PBEM Universe, can make itself convincingly female or male at need. No one knows if Masquerade even had a gender of its own when it was born.
Bodysnatcher, on the other hand, has long since abandoned his (or her) original body so long ago that she (or he) no longer remembers whether he (or she) was born male or female. The fact that the Bodysnatcher can't remember her (or his) real name doesn't help things.
The title character of The Saga of Tuck dresses in his suit to take a girl friend out only to be mistaken for a lesbian.
Jamie Carson, codename Heyoka, in the Whateley Universe. Looks and dresses very androgynously. This is made worse because Heyoka can gain the powers of spirits, and then physically shifts to look like that spirit, so Jamie has been extremely male (after getting the power of the bear spirit), and extremely female (after getting the power of a female earth spirit). Jamie has been beaten up because of this too, so it's not a good thing.
Flaky the porcupine from Happy Tree Friends. It's a long-standing, and still present debate due to her ambiguous appearance (unlike the other female Tree Friends, she lacks Tertiary Sexual Characteristics). Although Word of God from Flaky's voice actress in an interview says that Flaky's a female, there is still reason to believe otherwise.
Most Word of God states that Flaky is definitely a girl, but co-creator Ken Navarro loves trolling the fandom by playing up the debate for all it's worth.
It's later parodied in one episode where Flaky is shown unable to decide which bathroom she's supposed to go into.
The authors eventually settled the debate, as they constantly repeat in interviews that Flaky is indeed a girl, since they admitted they are tired of the trolling already.
In the SCP Foundation, Agent Diogenes has been exposed to so many magical artifacts that everyone has lost track of his/her gender, and Diogenes refuses to tell anyone. Despite this, Diogenes' psychologist is still romantically interested in Diogenes. Unfortunately, he Cannot Spit It Out and Diogenes is Oblivious to Love.
Sweet Tooth from Moshi Monsters. A running joke was that Sweet Tooth would get angry whenever their gender was asked about. There have been some slip-ups where they were accidentally referred to with a gender-specific pronoun, but many of these were edited out.
Worm has Circus, a mercenary who dresses as a woman in costume and has a male civilian identity. Even Tattletale isn't sure what biological sex Circus is or how he/she identifies.
Despite having a female body design Breeze Rider from Dusk's Dawn is voiced by a male actor and is most likely male. Still, it's never really made clear.
Fluffy and Uranus from Duckman. The title character's teddybear secretaries, they have distinct feminine voices, wear bows around their necks, and their behavior is mostly feminine, but apparently they have male genitals as they were humping a woman's leg in one episode and they say that they haven't been neutered. This is lampshaded in a later episode where the men and the women are separated from each other and forced to live on opposite sides of the city, but they can't decide on which side Fluffy and Uranus belong on so they make them crossing guards.
In Mission Hill, the gender of Carlos and Natalie's baby was never revealed, and on top of that its name wasn't mentioned at any point in the series; everyone called it "The Baby".
Despite being male in the books, Rusty was never referred to by gender-specific pronouns in the TV series and had a rather ambiguous-looking face and persona. This led to... interesting results when new writers were brought into the series.
In Shane Acker's 9, the twins 3and4 don't have any discernable gender. While they are 8-inch-tall automatons, the other ragdolls clearly indentify as male or female, 3 and 4 are never referred to as either.
In an episode of Lloyd in Space, the characters spend an episode trying to figure out the gender of a new kid named Zoit. When asked about it at the end of the episode, Zoit reveals that members of its (alien) species have no gender until they turn thirteen, at which point they have to decide to be male or female. Zoit chooses a gender, but we're never told which.
Although stated as female, Airrazor was physically ambiguous enough that when Beast Wars was dubbed for a Japanese audience, her gender changed.
The odd part is how the character was conceived. The toy was ostensibly, but not obviously, male. The character profile used male pronouns, but the show writers wanted more female presence. The blurb was rewritten and the character portrayed as female on the show. She was changed back to male in Japan because they thought it might sell more toys.
This got slightly uncomfortable for the production staff in the Japanese version, due to the characters' last minute kiss after two seasons' worth of hinting in the original English track. The characters in the Japanese version were played as a samurai and his ward, but when the moment came, they suddenly confessed, confusing a lot of 5 to 8 year olds who wrote in about it. Takara's only response was "some people are just like that." When a new series was adapted based on the show but following a slightly different continuity, they agreed to keep her female.
Also, Transmutate's gender was a mystery. Other characters mostly referred to the deformed Transformer with "it". Aside from that, there was one utterance of "she" as a pronoun, and Transmutate was given voice by the show's voice director and Transformers alum Susan Blu, leading to the prevailing contention the character was female.
On the King of the Hill two-parter about Buck's mistress' death, the mistress had a roommate named Gail. He/she looked and sounded pretty masculine, so people would assume he/she was a guy until they heard his/her name; however, it's never really clear if Gail is a guy with a Gender-Blender Name or a Bifauxnen chick.
In "BMO Noir" and a few other episodes, BMO has role-played as a little boy and had a fantasy noir adventure in thrall to a femme fatale. Neither of which negate the genderless determination but do call it into question.
In the episode "BMO Lost" Beemo displays more feminine traits, such as acting as a surrogate mother for a baby and "marrying" a comparatively masculine bubble.
In "Be More", BMO's creator calls BMO "he".
The caterpillar in Disney's Pluto The Pup short "Springtime For Pluto", who first seems to sing with a deep voice, which is later revealed to be a gag because it was underground and its voice was echoing through the tunnel, because once it emerges, its voice is very high-pitched and falsetto. However, during its Transformation Sequence, it again sings in a deep bass, though its mouth is never shown to move in sync with the words so it's questionable whether the caterpillar is even singing the song at this point. However since when it finishes its metamorphosis and emerges as a very sexylatinabutterfly, this could be a case of was femaleall along or a Gender Bender.
Alice from Superjail! used to be this for fans until the second season (and Word of God beforehand in a 2009 interview) confirmed her as being a transsexual female.
One episode of The Penguins of Madagascar had an annoying kid at the zoo informing Alice that the only way to tell a male penguin from a female was with a DNA test. The penguins laugh at his naivety until Alice declares that she only knows they have three males and one female, and "the birds know which is which." All four had been assuming they were all male, leading to a mass identity crisis as they tried to figure it out. According to their DNA test, all four ARE male. Alice is just uninformed.
Avatar Portal's Tera253 has a repeated running gag on whether or not he/she is male or female. Even after the user produced "proof" of his/her gender, it is still a common running gag that the user plays along with.
The Yu-Gi-Oh! real-world card game features the character Dharc, the Dark Charmer. There are 4 other monsters with similar names, all of which have been female, but Dharc could easily go either way. We're still waiting for someone to even ask the game's creator...
According to the French translation, it's a girl; the German translation opts to make it a boy.
One of PETA's mascots, a chick named Nugget, has been flip-flopping through genders lately. The site refers to it as male, and some early material with it shows it in a masculine role, but lately Nugget has been shown as a girl. His voice is childish and gender neutral too.
Babies. A lot of baby clothing can be gender neutral, and depending on age and how the parents dress them/cut their hair, it can be difficult to know if a baby or toddler is male or female.
The human fetus does not have a distinguishable gender for the first several weeks, during this period, short of genetic testing, sexing a fetus is impossible.
Hyenas are especially bad about this because of their rare matriarchal social structure, how the female is bigger and more aggressive than the male, and how their naughty bits are almost identical due to the female's usually high level of testosterone, to the point of making ancient people think they were Hermaphrodites or Gender Benders.
Birds. Usually, either the male is more flamboyant and colorful than the female, as with peafowl; or they look identical but the female is bigger, as with eagles. However, some bird species lack sexual dimorphism (and the genitalia tend to look identical, given that most male birds do not have an external penis), so the only way to tell some for certain is to get a DNA test. Sexing chicks is a full-time job in chicken hatcheries.
In ancient Egypt, due to the fact that Egyptian vultures have almost no sexual dimorphism, it was believed that all vultures were female, and as such, they were revered, because this in turn meant that vultures were all the result of virgin births.
Walruses are also a good example, since both genders have thick, bushy whiskers that look like beards. Even the babies have them.
As mentioned above, androgyne is a gender identity for something typically somewhere between male and female, or a mixture of the two, or sometimes something else entirely. Although not a requirement, many IRL people with non-binary gender identities strive to have an ambiguous presentation in order to feel as if they are accurately expressing their true gender.