The Great Mizuti, 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, they don't really have a gender. Guillo's personality is a gestalt created from the lingering memories of a man and a woman, thus is both and neither at once.
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).
Karma the Chameleon from Best Fiends goes by feminine pronouns in her bio, but her gender is also explicitly listed as "unknown", the only Fiend to do so. Some bios end up forgoing pronouns entirely for her as well, or even giving masculine pronouns instead, hinting at a genderfluid nature. Despite this, the book "The World of Best Fiends" lists Karma's gender as female, contradicting the website and game.
Leibniz of Blaster Master Zero II and its sequel is assumed male and spoken of with male pronouns, though character designer Natsume noted that he was given a slender neck and waist to appear more androgynous. The true ending of III has Leibniz unmask, revealing that she is a woman. Even after this, there was quite a bit of confusion regarding Leibniz's gender due to being voiced by a man (albeit one known for voicing female characters), Kane and Jason continuing to refer to her as male (due to not actually seeing her face), her apparent flat chest, and the fact that she could be mistaken for a very androgynous man. Inti Creates initially dancing around the issue didn't help. Ultimately it was laid to rest by Matt Papa, who revealed that Leibniz is indeed female. The image of her on the cover of the soundtrack of III is much less ambiguous, as it shows her wearing feminine (if a bit tomboyish) clothing, and she has a petite and feminine body underneath her armor.
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.
There are four possible gender settings: Male, Female, Neither, and Unknown/undetermined. You can also choose "Do not pester me with impudent questions!", a more roundabout way of setting it to undetermined.
You can choose not to have a gender preference for your mate, in which case the mate options' genders will not be specified.
In Chrono Cross, 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 P.E.K.K.A from Clash of Clans is so heavily armored, no one knows if they're male or female (or even if they're human). "Pekka" is a common male name in Finland, which is where the developers are based, but a loading screen tip refers to P.E.K.K.A as "her".
While Dark Choco Cookie has a very masculine appearance, official content goes out of its way to avoid using any pronouns for them, gendered or otherwise. Their costume's title, "Sovereign of Darkness", also uses a gender-neutral descriptor.
Cyberpunk 2077: You don't get to specify V's gender at any point during the character creation or gameplay. While V can blend different traits in both body (body type, voice, genitals) and style (haircut, clothes, jewelry), they are essentially treated as genderless throughout the whole experience, with only the player knowing what their V's exact identification is. There are mild dialogue changes (Dexter deShawn calling V "miss" or "mister") and it influences who V can romance, but that's about it.
In Dark Souls, Sif the Great Grey Wolf stirred some confusion due to their name (which may or may not be a Gender-Blender Name). The name Sif is a woman's name in most Proto-Germanic languages, which translates to the words "wife" and "bride". There is also a goddess named Sif, who comes from Norse mythology.
It's completely unclear whether the player character of Dark Tales is male or female. Some of the games suggest that they're male, some suggest they're female, and the rest make no hints either way. This is especially jarring since they're supposed to be the same character in each installment.
Deltarune's main character Kris is a Contrasting Sequel Main Character to the main character of Undertale, but Kris is still referred to exclusively with gender-neutral pronouns. They are called a "Momma's Boy" and their appearance in the World of Darkness riffs on classic takes on male RPG heroes, but it's still kept intentionally ambiguous.
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.
All the dogs in Dog's 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).
The eponymous protagonist of Dominique Pamplemousse is not only androgynous, but genderqueer as well, to the point they don't know what gender they identify as.
Dragon Quest V has Queen Ferz. Despite her "Queen" title, her monster form is a cyclops... which looks pretty male. It may have something to do with her power to cast illusions.
The unnamed blacksmith deity in the reveal trailer for Elden Ring by FromSoftware was assumed by some viewers to be two different people, since they look wildly different from different angles and camerashots, depending on the lighting. A full-body view of them from the back is mostly androgynous, with closeups varying between more masculine and more feminine.
You can play as a lady, a gentleman, or an individual of mysterious and indistinct gender. This last 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.
The Rubbery Men are only referred to as 'men' by convention (the player character occasionally gets an impression that one of them is 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 gender or their species. Both groups are commonly referred to as 'it'.
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.
Fate/Grand Order: The Chevalier d'Eon is extremely androgynous, having traits of both men and women but nothing definitive. The game states that one of the d'Eon's magical abilities is to change their sex whenever they please, and they seem indifferent when the player guesses their gender, whatever the answer is. The game's code considers them "genderless" for gameplay purposes (some abilities and events only affect male or female characters), with a couple of exceptions.
Shrouded in odd clothing ...is this a man...? ...a woman...? ...or should we ask...?
Quina from Final Fantasy IX (and by extension, most of the Qus, essentially a Genderless Race). The Lamias' "attract" attack works on them, so whatever they are they're attracted to women. Zidane won't protect them when equipped with the Protect Girls ability, though that could mean he doesn't know himself.
The Amalj'aa, which are a race of huge and burly lizards. The Amalj'aa are an interesting case since they do have males and females, but due to the fact that both sexes look and sound exactly alike and the race don't consider gender identity to be of any importance, it's quite difficult to tell who is what. Word of God says that even the Amalj'aa sometimes get confused with identifying each other, but when it's time to mate, their bodies release a unique scent that helps the race identify each other's sex.
Sylphs are another interesting case; they have two distinct subsets that kind of count as genders, in the sense that they define their reproductive habits and naming conventions, but neither could strictly speaking be classified as 'male' or 'female', and both types appear feminine by our standards.
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 how players would react to hitting a woman, so her gender identity was in flux for a while—her concept art◊ described her as a newhalf, a mildly derogatory term for pre-op trans women in Japan, while in other instances, she was referred to as a cross-dressing male. They finally settled on her being a trans woman in all continuities, the only difference being whether she's pre-op (Japan) or post (everywhere else that cares). Roxy, Poison's Palette Swap, was also listed as a newhalf; she's later described as a cisgender woman, presumably to distinguish her from Poison.
Nergal's morphs in Fire Emblem: The Blazing Blade are supposedly genderless. Gets applied to Limstella the most often, although Ephidel is probably just as ambiguous.
Mangle in Five Nights at Freddy's 2 is the only animatronic whose character doesn't have a generally agreed-upon gender. While all versions of Freddy and Bonnie are male, and all versions of Chica are female, Mangle may or may not be a different gender from its predecessor Foxy. Seeing as it's introduced as a barely-functional scrap heap of an animatronic, it's not easy to tell, and its intact Funtime Foxy design in Five Nights at Freddy's World has several elements of its design that conflict with either gender. Any other pieces of in-game evidence have been deliberately confusing. When the creator, Scott Cawthon, finally "confirmed" its gender, all he gave was a Mathematician's Answer. Many flame wars have arisen over the subject.
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.)
Very common in Guild Wars and its sequel. Most NPC races use the same model for both male and female characters, and most of the time you can't tell the difference until they actually speak. The ogres are the most egregious example, as all ogres have the appearance of horned, muscular male humanoids, but half of them, even the shirtless ones, are female. Even the charr and asura, two player character races in Guild Wars 2, use the same body type for both genders, though the faces are more masculine or feminine-looking depending on the gender.
Hylics has Dedusmuln, a vaguely plant-like humanoid who has an ambiguous body type and is never referred to with any pronouns. One NPC in the sequel is another member of their species, who also looks ambiguous and isn't referred to with pronouns, making it unknown if their species even has gender.
No one knows for sure what Seem's gender in Jak 3: Wastelander is. Their body type is ambiguous, which is amplified by the many layers of clothing and armor they wears. They could be male, because they were 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 their gender as male. 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.
In Journey, the mysterious robed player character has no gender.
Sabor is female in her source film, but male in Kingdom Hearts. It may be a journal error, but since she and her world have beenretconned out of existence, we will never really know. To make matters more confusing, Sabor is referred to as "it" in the novel adaption.
Three Dream Eaters, Meow Wow, Meowjesty and Flowbermeow. 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.
Kirby, the eponymous pink puff himself, is officially described as "gender unknown" in Japanese. He's often seen wearing masculine and feminine clothing interchangeably. He's more often treated as male (he has Ship Teases with the definitely-female ChuChu and Ribbon, uses masculine speech patterns when given full dialogue, usually refers to himself as "boku", has been referred to with the masculine third-person pronoun "kare", etc.), but the generally-accepted stance is that he is physically some form of Non-Human Non-Binary. In most localizations, he is strictly referred to as male.
Gryll, the final boss of the Japan-only Kirby's Super Star Stacker, is also not given a definitive gender. Gryll looks like a Cute Witch, but uses the first-person masculine pronoun "boku-chin", is not considered among the series' female final bosses, and has only ever been referred to as the neutral term mahoutsukai (magician).
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 note A bit of brilliance in retrospect; Canderous's native language of Mando'a rarely uses gender specifiers. As for Juhani? She was fully intended as Bioware's first Gay Option and much of her female-oriented dialogue stayed in during the first release, making her appear bisexual until the later patch fixed it and she was indeed confirmed lesbian. — which is canon, as confirmed by The New Essential Chronology. note This was a controversial call at the time, and so the Exile was canonized as female to placate the fanbase. BioWare later hung a big, fat lampshade on the whole thing by having the Revanites in Star Wars: The Old Republic state that Revan's gender isn't clear in the old texts.
In The Legend of Zelda: 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.
The Koroks in The Legend of Zelda: Breath of the Wild are androgynous creatures who are fairly interchangeable, and most aren't obviously male or female. Hestu◊ is the only Korok who is specifically male, not that you could tell by his appearance...
Len'en takes this trope and applies it to nearly every single character in the series. Out of a cast of over thirty characters, only one of them (a grandfather of one of the other characters) has a confirmed gender.
Vestera, a Vernal god/dess from Lusternia. Appropriately, their dominion is over illusions and dreams, so gender is less relevant anyway.
Mega Man 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).
Moshi Monsters has several characters whose genders just aren't revealed, but a notable example is Sweet Tooth-the last person to ask is still hospitalized and "wearing a gobstopper".
The Magypsies in Mother 3 are genderless, described as "neither human nor beast, neither man nor woman". They have rather feminine names and hairstyles, and wear women's clothing, but also have facial hair and masculine features.
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).
The dream characters of NiGHTS into Dreams are sexless, with gender down to the dreamer's perception. When voiced, NiGHTS sounds like a cross between a teenage girl and a little boy.
Not for Broadcast: It is unknown whether Alex Winston (the Featureless Protagonist), Sam (the protagonist's spouse) or Chris (the protagonist's spouse's sibling) are either male or female, as Alex is referred to as "you", while Sam and Chris go by "they/them".
Demon Lord Ninetails has no clearly defined gender in Ōkami. It's unknown if it is male or female. But considering it is a demonic creature with deific power, as well as being a shapeshifter that can take on male and female forms, it is likely genderless. Amaterasu has also stirred confusion, as she urinates with her leg up when using the Golden Fury ability. This confusion likely derives from the common misconception that only male canines can urinate with one leg up. This is more related to the degree of dominance a canine has, rather than its gender.
Niko, the adorable (and definitely not a cat) protagonist of One Shot. When asked if Niko was a boy or a girl, the developers replied "Yes, Niko is a boy or a girl".
Onmyōji: It is impossible to determine what gender Kohaku is, thanks to its androgynous voice and its using its name in place of a first-person pronoun. It is confirmed to be male.
This is a trait of the Lapine race in Pandora Saga; the race has no gender selection as 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.
Persona 5: Funny Animal Morgana, one of the party members, has the name of a legendary woman from the King Arthur myths, but uses masculine Japanese Pronouns and identifies as male. In both the English and Japanese versions, he's voiced by women performing childlike, somewhat-androgynous voices that the game itself describes as "boyish" (the rest of the party's voices are firmly described as "Boy's voice" or "Girl's voice"). Early in the game there's a dialogue option to guess his gender - saying that he's a girl causes him to get flustered and say he thinks he's a boy. The Maniax Handbook lists his gender as "???".
The protagonist of Pirouette is referred to as both a "daughter" and as a "husband" by various characters and has an androgynous appearance.
In Puyo Puyo and Madou Monogatari, Carbuncle's bios don't specify if he's male or female. He has Bust-Waist-Hip measurements in his Madou Monogatari Saturn bio, which is normally reserved for females, but it could have also been done as a joke.
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. They're a ghost of a male character, though.
Rakion: The Mage class makes it hard to determine if they are male or female thanks to their short stature, doll-like clothes, mask, and slightly shrill and raspy voice. Their Chaos Mode has them summon a puppet it rides on to fight, its deep and raspy voice heavily leans them into being male but there's no confirmation of their true gender as the game and the wiki refers to them as "it" instead of a pronoun.
TOMCAT, the expert hacker from Read Only Memories has no stated gender, and goes by the pronoun "them". Also, your Robot Buddy Turing has no gender identity either.
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 their gender.
The first thing Subaru from Sakura Wars: So Long, My Love 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".
In Secret of Mana, the sprite child Popoi has long hair and wears long robes. In the Japanese version, their gender is kept neutral, and they use the personal pronoun "oira", which is generally associated with masculine speech, but can be used by some women. The localized versions assigned Popoi a gender, but it varies on the language: in English, Popoi is male, while in German, they're female. The remake has Popoi voiced by women doing high-pitched childlike voices, and the redone English translation simply chose to use the genderless "they".
Zohar from Silhouette Mirage. He/She changes genders based on what powers they're using. Though, they're a computer program, so gender might be irrelevant.
In The Sims 4: Realm of Magic game pack, the NPC Morgyn Ember is marked as male in Create-a-Sim, however their preference is set to feminine clothes, they can't pee standing up, and they can't get anyone pregnant or become pregnant.
In The Spectrum Retreat, Cameron Worrall's logs are written in first person, and that, coupled with a first name that can be used by both men and women, makes their gender basically impossible to determine.
The cat-like imps in Cloud Spires in Spyro: Year of the Dragon all wear dresses, but their faces are masculine, and have gender-neutral names. The reimagining makes them even more androgynous with all except Fluffy sounding like preteen boys.
The Dwarf's gender in Stardew Valley seems to have been left up to player interpretation. Though a good chunk of the fandom appears to be guessing that they're male, no pronouns are ever actually used in reference to the Dwarf in-game. Their generic name and obscured face only add to the ambiguity.
Jamie, the player character's nemesis from Magical Melody, dresses in clothes that deliberately make them appear androgynous (they look 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).
Often times your child has no official gender, mostly in earlier games, so you can choose their gender. It doesn't affect the game, and the games don't refer to them using any pronouns. This applies to rival children as well in Harvest Moon 64, though one line by Popuri shows her kid with Gray is female.
Yutani from Subway Surfers is a kid in an alien costume. For a while, her gender wasn't hinted at aside for lips and eyelashes, until both the web-cartoon and bio confirmed that Yutani is female.
Arno from Swordcraft Story 2. Their gender is never revealed, and they refer to themselves as a "child of the wind". In the English games, their voice is masculine, but in the original Japanese it's feminine. The Japanese official site seems to suggest they're female by putting them 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.
In Sunless Sea, you decide how your captain's addressed, and gender-neutral options and portraits are available. The game also tends to actively avoid using gendered terms for your crew and your captain, too, so as to keep them ambiguous (and in your zailors' case, which pronoun is used is a tossup; your crew has male, female, and gender-neutral/unknown members). Finally, there are multiple characters whose given gender is never referred to, such as the Irrepressible Cannoneer and the Voracious Diplomat, and one overtly ambiguous character in the Alarming Scholar, who makes the narrator constantly struggle when referring to her (him?).
Sunless Skies, made by the same company and set in the same universe, is the same in this regard. The Incautious Driver is always referred to with neutral they/them pronouns. And they wouldn't have it any other way: when required to fill out a medical form, they resolve the ambiguity... by neatly crossing out the relevant field as not applicable.
Vivian from Paper Mario: The Thousand-Year Door is stated to be an otokonoko in the Japanese version, but due to the game's use of this term in katakana instead of kanji, it's unclear which precise definition this was intended to be, as the game's dialog can be taken different ways; the implication of Japanese otokonoko in popular culture, however, is essentially the equivalent of a male crossdresser (nevermind the fact that Vivian only seems to be wearing a hat). The English and German localizations skirt around the confusion by removing all references to it and referring to Vivian with exclusively feminine pronouns, while other localizations try to retain the nuance, with the Italian localization outright depicting Vivian as transgender.
The merfolk in Tales of Monkey Island. Guybrush is rather unsettled that he can't tell what gender they are. Winslow seems to be less unsettled and ends up in a romance with one.
Team Fortress 2: At first, given that Pyro had the same voice actor as the clearly male Spy, all indications 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". Word of God is "Not telling is funnier than anything we could say".
Tekken: Leo. Word of God says female and she gets a bikini in Tekken Tag Tournament 2 to affirm this fact. If one goes by voicing, though, it is hazy; Veronica Taylor provides Leo's voice during the time she is speaking English, but when she switches to speaking German, a male voice actor is provided. Her full name, Eleonora, is feminine. In Tekken 7, she can be customised to wear both male and female clothing, blurring the line even further.
While Touhou Project is well-known for its Improbably Female Cast, this wasn't established until the second game. The first game, Highly Responsive to Prayers, has no dialogue and no character profiles, leaving the genders of Konngara and Sariel completely up to speculation given their androgynous appearances, and SinGyoku makes things more confusing by shifting between a masculine form and a feminine form.
Kyros the Overlord, the triumphantEvil Overlord of Tyranny, is so Shrouded in Myth that nobody even knows their gender; it's noted that women tend to assume they're a woman, and men tend to assume a man. Then again, it's not even clear if they're a single individual, remotely human, or even real either. Only the Archons have met Kyros in person and therefore possibly know Kyros's gender (or in any case what Kyros actually is), but when they are asked about it, they always reply with "Kyros is mother and father." Or, in Sirin's case, switch pronouns randomly to screw with the Fatebinderfor laughs. It's worth noting that Tunon, Kyros' oldest and most devoted Archon, consistently refers to Kyros with female pronouns, though he gets annoyed if pressed on the matter.
In Undertale, Frisk's gender is unknown, and is referred to as "they" to preserve ambiguity; the same is true of the ghosts except for Mettaton (who seem to be genderless by default), Monster Kid, the River Person (who lampshades it when first met), and Chara. This is likely intended to enable players of any gender to relate to the player's avatar.
Monster Kid was originally referred to as "he" in the art book, though this was later edited. In the Japanese localization, they use the pronoun "ore", which is typically masculine, but not always. The rest remain ambiguous.
Onionsan's gender is also never stated, and many of the random monster encounters (such as Froggit and Whimsum) are usually referred to with "it" instead of gendered pronouns.
Versus Umbra: It's not clear what Bane's gender is due to them wearing a cape. The arena host even uses "his or hers" during a conversation with them.
Captain Viridian in VVVVVV is never explicitly assigned a gender. Even when giving (somewhat less-than-fulfilling) romantic advice to a trusted member of their crew, the issue of Viridian's gender is entirely unresolved in any capacity. The retro graphics do ultimately little to help clear anything up.
Xaku from Warframe is an odd example. While Xaku is referred to as "they", it could be referencing how they look like neither male nor female, thus being nonbinary. However, they're also comprised of multiple Warframes too, thus they could be both male and female and/or be multiple people. Either way, Xaku's pronouns are still "they/them".
World of Warcraft has the bronze dragon Chronormu/Chromie. Due to the dragonflights' Theme Naming any name ending in -ormu is male while females are -ormi, but whenever they assume a humanoid form it's always as a female gnome, and no other dragon has been shown shifting into a humanoid form of a different gender. Word of God later confirmed her to be female, meaning that for her species, she has a Gender-Blender Name and goes with a somewhat more feminine-sounding nickname instead.