Hong Kong. A normal and quiet Chinese boy or a very convincing Bifauxnen? The kid was eventually confirmed by Word of God to be male, but new fans still get confused...
Even China himself. Non-fans who know of the series' existence — or even occasionally some unknowing fans — call him a girl. Considering his long black/brown hair, his slim figure and small shoulders (at least in the webcomic), him being the second shortest of the main eight characters, his unisex human name, andhis female seiyu, it's not that difficult to fall prey to Viewer Gender Confusion when concerning him.
New Zealand is a source of this. Are they a cute short-haired girl or a simply effeminate looking boy in the vein of Poland and China? Time will tell...
Person 1: Are they lesbians? Person 2: They are two boys. Person 1: *mind fucked* o.o
In universe example in the English dub.
Soldier on seeing Holy Roman Empire's picture of young Italy: Oh ho! Is this the painting of your little girlfriend, or boyfriend, or gender-neutral chibi-thing?
Kugelmugel: a braids-wearing, art obsessed effeminate boy.
The Firey in Card Captor Sakura, with roughly equal amounts of evidence for both male (flat chest) and female (all the other elemental/humanoid Cards in the manga are female).
Considering the abundance of white-blonde Action Girls in Claymore, it's likely that most fans assumed that Isley was yet another one until he took off his shirt. Of course, you can hardly blame them — does this◊ look like a man's face to you?
V.V. of Code Geass. A young boy, voiced by an actual young boy (for once), whose blond hair (longer than his body) and androgynous name throws many a viewer off.
Ed from Cowboy Bebop is just so tomboyish she easily passes as a boy. The fact that the Hungarian dub gave her a BOY's voice doesn't help. The crew apparently didn't even realize it until Faye shouted it out at the end of the episode she's introduced in.
Manga readers often presumed Mello female by his tight pants and haircut. To combat this, the anime gave him an uber-masculine voice — but then gave Near a female voice actor. In his first appearance, Mello wears what appears to be a dress, tall boots, and a fur-lined coat◊ longer than the "dress" (in actuality his vest blending in with the shadows of his pants). He's also got some very distinct hips in some shots, and at least one manga cover even made him look like a flat-chested girl. One moment in the anime also gave him some rather unfortunate shading on his vest, making him look like he had small breasts.
Even worse than that is Rem, Misa's shinigami. Sure, shinigami only have Purely Aesthetic Gender, but it's still a bit of a brainbreaker to realize Rem is a female. It doesn't help that her voice is extremely hoarse.
Digimon: This is a problem for any digimon who doesn't have any overt secondary sex characteristics, including the very young (like Poromon or Calumon) or ones who look very different in certain evolutions. Usually, voices or Tertiary Sexual Characteristics make it clear once more forms are shown.
Digimon Adventure has Garudamon who has been suspected to be male despite the other forms being obviously female, just because of the body shape (more anthropomorphic the others, long blonde hair).
Hawkmon from Digimon Adventure 02 mainly because he's partnered with a girl and all the other children get Digimon who are their own gender. Miyako/Yolei seems to be the only Digidestined to have a Digimon who isn't the same gender as her.
Cutemon is Xros Wars's character of questionable gender: it's pink, wears floral-printed headphones, and has a high-pitched voice. And it's...a boy. His use of boku gave him away.
Frieza/Freezer from Dragon Ball Z — in Japanese, he is using fairly feminine language. In order to try to approximate this in English, where words are the same regardless of the gender of the speaker, they had him voiced like an elderly woman, which made him come across as a hermaphrodite. It didn't help that he had purple lips that looked like he was wearing lipstick, either, or that his final form had no genitalia. His other forms, however, did have a black speedo.
Bedivere in the anime of Fate/stay night. The guy's just as feminine as Saber and has Mamiko Noto's voice. What are we supposed to think?
Ladios Sopp in The Five Star Stories, with his shy and soft-spoken demeanor, effeminate voice, baggy yellow clothes, big dewy eyes, and long braided hair is constantly mistaken for a woman. In fact, one character, Voards Viewlards, eventually learns the truth and still flirts with him! It doesn't help that Sopp is the alter-ego of the king of the A.K.D., who is named after the ur-goddess of Japan, and frequently played by a female Body Double while the king is off playing as Sopp!
Envy counts in the manga version of the story. He's stated in an omake as having no true gender, which means that it may or may not be a canon statement.
Wrath from the first anime is of similar circumstances to Envy. He's young, has long hair, a female voice actor and the fact that he's lived in an isolated island cause confusion as to his gender.
Den, Winry's dog, is female but is constantly seen as a male thanks to her unisex name and the fact dogs that look like her are typically male in fiction.
The anime series of Gantz has the shorter of the bum-hunters (Hajime Moroto, which isn't much help for most English speakers, and you don't find out his name until after his partner in crime spills the beans anyway), whose elfin features, just-under-shoulder-length haircut, and bulky coat makes his gender ambiguous at best until the larger bum-hunter is talking about him to someone else and uses the male pronoun (this doesn't occur until several episodes after the characters first show up).
At first glance, Kazuki, with his long flowing hair, long lashes and slim frame, would mislead anyone into thinking he was a girl. It didn't help that he was portrayed wearing female clothes in various points of the manga and anime, and he's grouped with the girls in one of the ending clips and some of the official artwork (apparently, the writers were in on the joke). He is also a victim of Tokyopop's She's a Man in Japan translation.
It's quite justified in the manga: his martial arts style requires flexibility, which is trained by having him spend his early years wearing female clothing, and learning feminine skills.
Kazuki has a female counterpart, Ren; she appears at first glance to be male, to the extent that the other characters don't realize she's female until she points it out (read: yells it) in passing. Her personality doesn't help much, either... nor does her previously freaking out after realizing that the person she kissed to deliver a medicine was male (said person being... Kazuki, naturally).
Shido: That's a girl? (muttering) I suppose if Kazuki can be a guy...
An interesting case comes from Girls Saurus. That cute girl getting molested by men on the train and is saved by the main characters? Yeah, that's a guy. But wait, he's got breasts and is wearing a skirt!... Not really: turns out that he's so feminine that the main characters (and, indeed, everyone who meets him, man and woman alike) hallucinate that he's built and dressed like a girl.
Seguchi Touma of Gravitation is feminine enough to pass for a woman for several episodes — even though he was always referred to as male in the subtitles and he had been revealed to be married to Yuki's sister!
Tieria Erde. Likely the first response of most viewers the first time he speaks (in a rather masculine voice) is "That was a guy?!" This spawned a bit of a Memetic Mutation in Japan, as many artists gleefully went right to work in creating pictures of Tieria as a girl... While only having to slightly change some visual details (and in a few cases, giving "her" a somewhat Genki Girl personality).
As if Tieria wasn't bad enough, season two brings a new character named Regene Regetta, who looks almost exactly like Tieria (curly hair aside) and has a very feminine voice (and up until his character design was released shortly before the season started, there was rampant confusion and speculation over his gender. Then again, his aforementioned girly voiceis provided by Romi Park, of all people).
ALL Innovators are basically this. Just try to guess the genders of Revive Revival or (really worst offender here) Hilling Care. Hilling was wearing a tuxedo in Ribbons' party and looks rather boyish, but still uses pronouns and speech patterns that are distinctively feminine. Word of God is that they do have physical sexes, but most of them identify as genderless.
Kuu and Kana from Haibane Renmei, helped by the latter's Wrench Wench status. Even the manager of the cafe where Kuu works thinks she's a boy.
A number of those who saw the first Hellsing anime expressed annoyance that the titular Sir Integra was such an embarrassingly pretty Bishōnen. This is somewhat excusable, what with the author referring to her as "Sir", presumably due to a misunderstanding of British titles. A woman given the honor of joining a knightly order is referred to with the honorific "Lady" or "Dame" (depending on the order and rank). It was explained somewhere that it is some sort of a legal fiction — to give Integra a chance to inherit the Hellsing name and Round Table position, she was officially registered as a man. So being legally a man, she is entitled to a male honorific.
Warrant Officer Shroedinger looks very much like Victoria Seras but with cat ears.
Fujiwara no Sai from Hikaru no Go. It's not so difficult to see what his gender is if one has a passing knowledge of Heian court dress, but the western audience is left wholly at sea.
The Swedish manga Bleckmossen Boyz features a character named only as "Alex", whose deliberately unspecified gender and androgynous looks become a Running Gag. One of the characters eventually goes so far as to look up his/her full first name, but finds it to be "Alexis", a unisex name.
Jakotsu of the Band of Seven is a prime example of this, confusion deriving from him being a cross-dressing homosexual coupled with the fact that his voice in both languages is provided by a woman. Apparently creator Rumiko Takahashi originally intended for him to be female, but opted for making him gay instead, as she wasn't keen on the idea of having InuYasha fight a human woman.
Shippo, the fox demon of Inu-Yasha's group is often mistaken for a girl due to having female voice actors and wearing a bow in his hair.
There was also a filler in which a sibling of an early set of antagonists, the Thunder Brothers, challenged Shippo to a fight for revenge against "his" Brothers. Both actually sound feminine but they're young kids and the practice of hiring women to voice kids is very common practice. At the end of the episode, it's revealed that the younger "Brother" was actually a "sister"!
Sesshomaru, in the early chapters of the manga, tripped up many people until Inuyasha explicitly referred to him as "Brother".
Arion Rosemariné has confused some scanlators. He dresses entirely in Gorgeous Period Dress, has long blond Ojou Ringlets, and is voiced by a woman in the OVA. The only way many people have realized his actual gender is through the fact that he lives at an all-boys boarding school. In any case, his gender is proved once and for all when it's revealed that Auguste raped him, and Auguste only goes for pretty boys.
Liliath Florian manages to be an even easier to get confused about. This minor character has flowers in his hair, is very effeminate both in appearance and personality, and seems to be flat-out referred to as a girl by other characters... and yet he goes to the very same all-boys school.
In the Philippines, Kurama initially had a distinctly female VA and started out female. Yusuke was surprised to learn he was a guy on their first assignment in the demon world. The explanation given was that he was male in the demon world but female in the human world. However, all later appearances had him strictly male, regardless of which world he was in. The dubbers were apparently also confused as to Genkai's gender as the name they gave her was Jeremiah and her VA was very noticeably male. It wasn't until after she first transformed into her younger self during the Dark Tournament that she was given a female VA.
There is also the one shot villian Miyuki a very feminine looking demon dressed in feminine clothes with an equally feminine voice, after knocking him unconscious Yusuke and Kuwabara pull down his pants and are shocked that such a feminine demon is actually male.
Many first time viewers of Keroro Gunsou think cutesy, shiny-eyed Private Tamama is a girl. Oh how wrong they are...
Kino from Kino's Journey causes this trope. It's not helped by a certain tendency to use both male and female pronouns on and off. Thankfully, the real gender is a plot point, and a good bit of the character's development and relationship with the audience is developed around the confusion... even if most viewers get it wrong the first time. The English translation of the first novel moves The Reveal chapter to the start of the book to avoid having to dance around pronouns, while the English dub of the anime gives Kino something of an androgynous voice, so you're not sure if she's a boy or a girl for several episodes until a gender pronoun is used.
In the Koudelka manga, the little boy turns out to be a little girl whom her mother, a prostitute, disguised as a boy to save her from being used as a prostitute in turn.
Kurau from Kurau Phantom Memory. Even though she has an alternate persona in Christmas, who is obviously female, her ambiguous clothing, hairstyle and seiyuu cause some confusion. It gets better with age, though.
There is some disagreement about the gender of Pierre◊ in Kyouran Kazoku Nikki, which may well be the goal of the author of the original light novels. Pierre's real name, Akeru, is a typical girl's name, although there are no truly fixed rules for that in Japan. Also, Pierre has long braided hair and female mannerims, but in the world of manga and anime (and outside of it) that doesn't have to mean much. Pierre furthermore uses the gender-neutral "watakushi" for "I", which complicates matters even more.
In Magical Girl Lyrical Nanoha Striker S, thanks to her deep voice, baggy clothes, and boyish looks, a lot of people mistook Otto to be The One Guy amongst the Numbers Cyborgs. It wasn't until Jail revealed that all of his cyborgs are pregnant with a perfect clone of him did those people realize that, yes, she's a female too.
The first example would have to be Haku. In their first interaction, Naruto thought he was a girl until Haku stated that he was a guy and even then commented that "he's even prettier than Sakura-chan!"
To add on more gender confusion, Haku is voiced by a woman in both the Japanese and every dub version of the anime!
Gaara's uncle Yashamaru looks exactly like his sister. Even though the "-maru" indicates a male, some translations referred to him as a female.
For that matter, had Sasuke not mentioned that it was a "man" that he vowed to kill or had he not been introduced as Sasuke's "brother", many a fan would've assumed Itachi was a girl at first glance. The luxuriously thick eyelashes, long hair, and short and slim build (when seen next to Kisame) didn't help. In a flashback to the night of the Kyuubi attack, the only feature distinguishing him from his mother's established appearance was the facial lines he inherited from his father.
Deidara's gender was ambiguous at first, as the long blond hair and slim build suggested a female. It doesn't help that he looks like Ino's older sister.
Sai's midriff exposing top and slim build, along with his Emotionless Girl status cast some doubts about his gender at first.
Taken to its extreme with the seven-tails host, who is only seen on the splash page of one chapter and an artbook where the author himself stated he was unsure of his/her gender. He eventually settled on female.
Kurenai's daughter in chapter 700 caused this reaction with a lot of fans.
Inojin, Ino's and Sai's child, is male but has soft features and a ponytail. He gets his androgyny from his dad.
Yoshiyuki Sadamoto admitted straight out in an Omake to an early volume of the Neon Genesis Evangelion manga that Shinji's design is basically Nadia's with a different haircut. So this is understandable. Not to mention that his Japanese voice belonged to a lady who is very well-known for her deep voice. Case in point, she's also well-known as Sailor Uranus, who has her own entry.
In the manga of Paradise Kiss, Yukari IDs Isabella as a cross dresser virtually as soon as she meets her. However in the anime this doesn't happen, and no-one makes any reference to the fact that she's actually a biological man until episode 10. Consequently, watching the anime first means there isn't even have a clue that anything is amiss until that point- given how fruity most of the characters were, there didn't seem to be anything odder than usual about her, especially as she's voiced by a woman in both languages.
Epsilon from Urasawa's Pluto. What makes it even more difficult is that he's based on the Astro Boy character Neptune (who is male), but shares the name and character traits (such as being a Technical Pacifist) of a female character from the same series. It doesn't help that the fan translation has referred to him as "she" once or twice...
In Ranma ˝ you have Konatsu the "Genius Kuoichi", a crossdresser who managed to trick Ukyo and the rest of the cast into thinking he's female until his clothes get blown off.
A lot of fans are under the impression that Team Rocket agent Hun was given a gender change in the dub despite the fact that the script directly calls "her" a man at one point. It really doesn't help that his dub voice sounds makes him sound like a woman.
Shaymin in Giratina and the Sky Warrior. Cute little flower hedgehog... forme change to kickass doggy-reindeer thing. Coincidentially, its dub VA is well known for voicing Tails in Sonic the Hedgehog games and Sonic X.
In the Adventures manga, Gold ends up mistaking Bugsy for a girl on first sight, and even flirts with him.
Another Bugsy incident happened in another manga◊, Golden Boys. Their Gold had mistaken Bugsy for a girl but it was a much milder example and was dropped instantly. It doesn't help that he's a Bishōnen — cute little boy mix in that manga.
Ash's Pikachu gets this a lot. It's male, but wasn't confirmed this until the infamous Diamond & Pearl episode with Rayquaza and an evil Togepi.
Most recurring Pokemon have confirmed genders, but are almost always referred to as "it".
Kenshin from Rurouni Kenshin. He has long red hair, a slim build and a rather feminine face. The author, in fact, stated that he gave Kenshin his X-shaped scar to make him look more masculine. Not surprising, as his Real Life counterpart, Kawakami Gensai, was said to be of a small stature, slim build and unusually pretty... for the deadliest assassin on the Loyalists' side. Both he and Kenshin had their fair share of being mistaken for a woman — and the latter in-story too.
Zirconia is a decrepit old woman who's initially only seen in shadow, so by the time she was given The Reveal she had already been dubbed as an old man.
Sailor Uranus has occassionally run into this in some adaptations; while she has even occasionally portrayed as female only while transformed, many adaptations find hiding her gender too difficult or ingenuine to bother with.
Fisheye is an even worse case, being a Wholesome Crossdresser. Even some dubs that didn't have much problem with crossdressing and homosexuality to begin with managed to get his gender wrong.
Andromeda Shun has a feminine voice in the Japanese dub, wears pink armor, has a slight figure, and is prettier than most of the female cast. In fact, most of Saint Seiya's Bishōnen are prettier than most of its female cast, and have fooled many unwary viewers. It confused even the French dubbers who had a female voice actress dub Shun's lines in the first few eps, until Ikki calls Shun explicitly his "brother". And later, there is this episode stating that female Saints wear masks, but the confusion's still there.
It's even worse with Pisces Aphrodite (also given a female voice in the French Dub). Even knowing that female saints are supposed to be masked, and thus he has to be male... he's girlier than Shun, wears lipstick and fights with roses... and seriously, he's called "Aphrodite"! Saga even describes him as "the warrior of beauty".
Aries Mu, possessor of a very pretty face and long, shiny pink hair, also got a female voice in the French dub.
Aquarius Camus was — in the manga — explained to have long red hair, long red fingernails and feminine features because the Aquarius sign represents the myth of Ganymede and has been seen as both male and female. This didn't transfer into the anime, but it left viewers wondering exactly what other excuses Kuruamada had for all this Ho Yay and Viewer Gender Confusion.
The queen of this trope is Lacerta Misty. Big poofy blonde hair, wears pink, wears pink "lipstick" and says "alrighty"? It took until he was completely naked for most of the viewers to realise his gender — and even then some didn't believe it. Even when his dub voice is none other than Vic Mignogna.
The BL light novel and anime series Sukisho has Team Mom Nanami. Everything about him is completely feminine and he even goes by a female name. His clothing when outside or at work are rather ambiguous, but at home he's usually seen wearing an apron or bunny slippers. The only hint on his male gender ever are the pronouns in the subtitles and in one episodes even the subtitle refer to him as female.
Quite a few people mistake Fai from Tsubasa Reservoir Chronicle and his "poofy Toma Seguchi coat" for a girl. Even though he is most definitely male, no matter how good he'd look in a dress.
Canute from Vinland Saga amongst both the fandom and in-canon is confused for a girl when he first appears. He's male though, most certainly.
Midriff-exposing, bangle-wearing Toboe from Wolf's Rain apparently caused author gender confusion — an artist's note accompanying character model sheets in the first manga volume jokingly(?) laments "I thought he was a girl... sniffle".
Bakura from the second anime Yu-Gi-Oh!. officially, he's a male with long, flowing white hair and a voice in the original Japanese that leaves everyone wondering. It doesn't help when you find out that he actually had a female voice actor.
Amelda/Alister has a feminine name in the original Japanese. And he looks a lot like a tomboyish girl with a small bust. He seems like a Dark Chick version of a Shorttank, except for the fact he's actually male.
Doctor Crowler. His voice in the original is very clearly male, but the dub does add quite a bit of confusion to the issue. The purple lipstick doesn't help. The dub even lampshades it from time to time.
Even worse is Yubel. You're pretty much guaranteed to guess the wrong gender. Because "she" and "he" are both used in reference and Yubel is technically a hermaphrodite at this point. Even the original Yubel is very androgynous, though not to the same cheater level as most of the series.
Rally is supposed to be a boy, but has long red hair, huge round eyes and a voice that could be confused for either gender. His clothes don't help.
Jeager is short with a slim build, a feminine high-pitched voice, lipstick and pointy ears. He's supposed top be a clown, but his gender is not very clear.
Luciano is even more confusing. If he wasn't wearing a male uniform, the long red plait and girly voice could fool anyone. It certainly doesn't help those who play the World Championship Series games that, in the 2011 title, Luciano's hairstyle and outfit are only available for female characters.
Ranewater Calder from Zombie Powder. It didn't help that he's the leader of a gang called the "Ash Daughter Gang", and it certainly didn't help that he was drawn as being much prettier and feminine than pretty much all the women in the series.
Husky, the fish-boy from +Anima. It even gets to the point where the other characters in the manga are confused about what his gender is.
He even has to drop his pants at one point to convince Nana he wasn't a girl, since she refused to believe anything he said to the contrary.
Yuki Kaori has been guilty of making her characters (of either sex) look more like the other's gender thanks to her love of androgyny. She mentioned once about the character Rociel from Angel Sanctuary that she got letters early on asking if he were male or female. Her response was something like "Well, I did say he was Katan's father..."
Speculation on the Youtube comment boards for Glass Fleet continued to wonder why androgynous Michel was referred to as a boy but obviously sounded like a woman. Once that was cleared up, the fans turned to poor Ralph, a child repeatedly referred to as a boy, with a boy's name, but with very feminine mannerisms and an obsessive crush on an older man.
After Art Evolution, Katekyo Hitman Reborn! has suddenly started getting a bunch of them. Chief offenders include the Acrobaleno Viper from the Varia and Kikyo and Daisy of the Real Furneral Wreathes. Only in the latest chapter has the translator been certain that Kikyo is male and no one can is yet certain if Daisy is this or a Bifauxnen.
It's now been confirmed that Daisy is a boy.
Zaji of Letter Bee is thought to be either a boyish-looking young woman or a man. It doesn't help that early on in the series, a girl named Nelli who looks a little like Zaji is mistaken for a boy.
Waq Waq's Yoki looks like a woman with a feminine face and long hair, and dresses in loose robes, but is referred to as "he".
Several Saki viewers were quite surprised when Jun shouted "I'm a girl!", partly thanks to her heavy usage of the "ore" pronoun. Guess they just assumed that she was The One Guy in the entire tournament.
Well, she does wear pants combined with a skirt, but perhaps that's considered men's clothing in Japan.
Although Luppi Antenor was clearly male to a lot of the fanbase and very into Rangiku's sexy body, he caused a lot of confusion with parts of the fandom who felt he was looking and acting mostly like a woman. The anime helped that portion of the fanbase by giving him a very masculine voice.
Some people have mistaken Wonderweiss Margera for female.
Berenice Gabrielli, a quincy killed by Kenpachi Zaraki. The fandom cannot agree whether the character was male or female, and Kenpachi's comments about this quincy are gender-ambiguous in the native Japanese, leading to translators and English-speaking fans to debate the subject. The flashback images of the face offer very few clues, as the character is either female or bishounen and could go either way. The feminine name also offers no clues as Kubo has given quite a few of his male characters feminine names, including the main character. When Viz translated the text for the official English release, they made the character male.
Another Stern Ritter, Äs Nödt, also caused much confusion since appearing in the manga. This is thanks to having big girly eyes, a hairdo resembling Godiva Hair, a concealed lower face region, and a big figure-hiding cloak. The character has been pretty much confirmed male, however.
Ill Girl Kayoko Nagato in Ojamajo Doremi has a name that isn't very girly, an androgynous voice, and a very short haircut (her dinosaur tooth necklace doesn't help). The only telltales was her pink backpack and Doremi using -chan when referring to her.
Several characters from the Loveless manga. Yayoi, Kio, and both halves of "Zero" who have longer hair in feminine styles, feminine body structures, and most often baggy/feminine looking clothes. And no voice actors to seek guidance from.
At least in the anime, Yayoi, Kio and Youji are all voiced by blokes, and even Natsuo's seiyuu is best known for voicing young boys. The real prize for this trope should go to Yurio, who appears quite late in the manga, has a very girly hairstyle (bobbed with long curls underneath), has the kanji for "man" in his/her name, and generally gives people a headache as to what his/her actual gender is.
Nachos from Magical Nyan Nyan Taruto is actually a boy, but it's very hard to tell until his gender is actually stated because he has long hair in braids and looks more feminine than his sister, Chips. Their gender-neutral names don't help, either.
In the English dub, Crona is referred to with male pronouns because the dubbers didn't want the dialogue to sound too awkward by avoiding the subject. No one, especially the ones behind the English dub, has a clue behind Crona's gender.
Some people that read YuYu Hakusho may have mistaken Kurama for a girl, due to the hair and the way that his shirt is drawn (with a fold at breast-level), only to be embarrassed when they find out otherwise. They should feel better when they read the story which has him crossdressing to investigate a haunting at a girl's highschool campus, and several people mistaking him for a girl even when dressed normally.
The Hungarian dub of the anime has a deep but definit.
Hermes from Part 6 is really masculine. If not for text and the fact she's in a female prison and some of the insinuative events at her first appearance (and other times), one would have sworn she was a he.
Also Narancia from Part 5, but in reverse as thinking 'he' was a 'she'. His attire and Japanese game's voice actor doesn't help.
Buccelatti is another one from Part 5 — his pageboy haircut and fabulous outfit being mostly to blame.
Kira from Legend Of Himiko can definetly throw off viewers. The fact that her/his higher-ups call her/him "Lady Kira" assure us that Kira is female, but her voice does sound like that of a boy who sounds like a girl.
Haihane of the disciplinary squad in Sekirei looks like an androgynous man until she loses her jacket (revealing she's female).
Junior in ROD the TV. Letting down his long, flowing hair, after being introduced in boy's clothes has a tendency to cement the confusion,and don't forget Maggie who even does not wear a dress to a party or ball.
In Trigun, when you first see Vash and Knives as children they look very much like girls, but once you hear them speak you realize their gender.
Rei of Saint Beast. Especially when the viewer doesn't know the show belongs to the BL genre.
Plenty of people have mistaken Asuka from Zettai Muteki Raijin-Oh for a girl due to his effeminate appearance and teeny-tiny shorts, even on this very wiki. His name doesn't help, as it's the name of a female pilot in another, more well-known series; and that his voices in both Japanese and English versions are pretty androgynous. Not that his fanboys care...
While revealed by p. 8 in Volume 1 of the manga, Nakahara Tatsuki from Boku No Tsukuru Sekai, a very convincing Wholesome Crossdresser and Magical Projectionist teacher, is revealed to be a man from his son Ritsuki when he calls Tatsuki "Dad."
Norio Koga in Naru Taru. Apparently, some readers have gone through the entire manga without realising that the character is (biologically) male. It doesn't help that his hair is nicer than most girls, and that he's lusted after by both male and female fans.
While not the most common, Chopper from One Piece in a few cases, being the Ridiculously Cute Critter with a female VA and pink as his main color. On the Drum Island arc commentary, some of the dubbers at Funimation admitted they weren't sure of his gender until hearing his scream about being a man.
Then there's Dellinger, who, though flat-chested, acts flamboyantly, has rather shapely legs, and wears high heels. As of this writing he has not been specifically gendered in the manga (at least in English translations), and is drawn rather ambiguously. The anime, however, seems to want viewers to make no mistake that he is male.◊
D.Gray-Man has a lot of bishonen, some of whom reach this level of girliness. Kanda, for example, is easily mistaken for female at first glance due to the long hair, and he's got nothing on the (male) Creepy Twins Jasdero and Devit; both are rather ambiguous-looking, and in combo, well◊... The author actually had to clarify their gender through the Fourth Wall Mail Slot.
Baka Test's Hideyoshi Kinoshita. It's basically the point of the character. It's gotten to the point that the fanbase has considered Hideyoshi to have be its own gender. Also to mention is that he can emulate anyone's voice.
Heroman gives us Joey, the main character. While his name is obviously masculine, his design is androgynous at best and his personality, body language, and especiallyhis voice are much closer to a tomboyish girl than a teenage male. It's enough to make you wonder.
Thick shoulder-length blonde hair, girly voice, short, thin, even a little curvy... Yeah, I doubt there's anyone who knew he was a guy when they saw him for the first time.
Powerpuff Girls Z's version of Snake. It doesn't help that the English dub of the series actually did turn him into a girl.
Risky from Risky Safety is androgynous, a voice that sounds like it either belongs to a tough girl or a young boy, and...ah, it's just hard to tell. The japanese version never explicitly came out and said anything until about two-thirds of the way through, and even the dub got confused, referring to the revealed-female Risky as male, calling her "him", "he", and having a minor character refer to her as "Mister Shinigami." She also refers to herself as "ore was" in Japanese, adding to the confusion.
Huang Chaun from Riki Oh is a very effeminate man with a slender build, lipstick, long eyelashes, and long painted finger nails, but he has a rather masculine voice in the anime.
In the live-action adaptation, they went further and had him played by an actual woman and voice-dubbed by a man.
The Headless Rider in Durarara!! is ambigious for the first two episodes, as she's only shown from angles that mostly hide her feminine curves, and is referred as male by the people who talk of her as an urban legend. Then it turns out that she was the female narrator all along.
Ryo Kuromatsu from the manga The Magic Touch is a girl but she looks remarkably like a boy and is rarely seen in her school uniform. When first encountered in the manga, her gender is not specified, save for one character calling her "she" in order to surprise Amane Mihime, and the reader who didn't catch the gender drop.
Hansel and Gretel in Black Lagoon. By the end of their story arc, it's still not very clear if they were brother and sister, sisters, or brothers.
Takumi in Gad Guard. He has long blonde hair styled into a hime cut and appears to be romantically linking up with Hajiki in his first few episodes. However, switching to the English-language dub leaves the viewer in little doubt as to his gender as the voice sounds masculine (despite being voiced by a female in the English dub).
For people who have not seen Seirei no Moribito from the start, it may be a surprise that Chagum is a boy.
Neon Genesis Evangelion: Occasionally happens when Maya is mistaken for Shinji and the other way around. The source of this confusion is that, Word of God, Shinji's character design is actually a female head slapped onto a male body: he's based on Nadia, the main character from Gainax's previous series, "Nadia and the Secret of Blue Water", just with shorter bangs and the jewelry removed. So when people accuse Maya of looking "butch" its actually the other way around: Shinji is based on a girl. Maya is just how Gainax draws generic girls.
It happens a lot in the Alternate Continuity Gakuen Datenroku ("Records of Heaven's Descent"), where Shinji is at his most androgynous and really looks like Maya and where it's sometimes necessary to have a close look to realize that the cute short-haired girl is actually him.
It does not help that any genderswap art of the cast basically just switches Shinji's and Maya's uniforms.
Shinji is voiced by a woman in the Japanese version, but voiced by a man in the English dub.
Magical X Miracle inspires a lot of this. For starters, the main character Merleawe is a girl who looks exactly like (and must pretend to be) the male Master Wizard, who went missing. In the bonus art, it's really hard to tell which is which. In addition, several fans have expressed confusion at Yue's gender. He's actually a man, but between the Rapunzel Hair, the Qipao, and the Gender-Blender Name, things can get confusing. It gets more confusing when, in book 2, a female character who closely resembles him (both in looks and Sugar and Ice Personality) was introduced in Glenn's flashback. Perhaps Lampshading that, later in the book, General Lenolora makes fun of himfor looking like a girl.
Hana from Michiko to Hatchin; she even got mistaken for a boy in one episode. She's young (around ten years old), dresses in a boyish manner, and her voice isn't all that feminine. The series takes place in South America, so Japanese Pronouns can't help either in-series.
Beyblade gives us Oliver (at least in the English dub). With his femimine features, girly voice, pink beyblade, and unicorn bitbeast, many people assumed he was female. Some of them even thought his name was actually Olivia, and was just being mispronounced. Eventually everyone got the hint when he was continually referred to as a 'he'.
Beyblade's spinoff, Metal Fight Beyblade, has quite a few guys with questionably feminine appearances. Thankfully, most of them are saved with masculine voices.
Wandering Son is complicated due to it being about Transsexuality and crossdressing. For the longest time fans thought Makoto was a Camp Gay boy until she was confirmed as mtf in high school chapters.
Gamaran has Ranmaru Itou, a male samurai who looks very feminine.
The main character from Chi's Sweet Home is female; this trope is easily justified since she's a two-to-three-month-old kitten. The verdict is out on her new kitten pal Cocchi from the manga and OVA though; the Japanese version has him/her refer to him/herself with "ore-sama", and the overall interpretation leans toward male. But you never know with kittens, even in Real Life when sometimes you have to check twice.
Un-Go has the detective's assistant, Inga, who apparently is an energetic young boy, although it's hard to be sure. The main issue being that for yet unknown reasons, possibly provoked by how close the 'truth' is in a case, Inga transforms into a vaguely monstrous (it's somewhat feline, and looks to be bigger than a person should be) female who insists to Yuuki that she be told a single question to solve the case.
In The World God Only Knows, Chief Dokuro is typically assumed to be male, despite the fact that all New Devils are female. This extends to the anime, where in the dub she has a male voice actor, and in the sub her voice actress uses such a coarse voice that everyone assumes she's male.
Ana Graham from Phi Brain Puzzle Of God is apparently a dude despite looking, dressing and sounding like one. Apparently confirmed after taking his shirt off you only see his back.
Jun from Persona -trinity soul- is very easily mistaken for a flat-chested girl. Slightly justified in that his twin sister died in a car crash and donated part of her brain and organs to him, resulting in a shared Persona.
Leeron from Gurren Lagann can easily be mistaken for a woman at first because of his feminine look and crossdressing nature, but it turns out he's actually a homosexual, crossdressing man.
Many characters from Inazuma Eleven but Kirino Ranmaru◊ from Go stands out. He has long, pink hair which he wears in pigtails.. But is nevertheless a boy. And a few players have been confused about Kazemaru and Miyasaka's gender upon first playing the game. Sure, the player will figure out Kazemaru's a guy after hearing his voice, but Miyasaka looks and sounds so girly in the anime that one has to constantly remind themselves that he's male. Probably justified in that they're young and might not have hit puberty yet, but come on...
Tower of God could easily have the title Gender Confusion: the Webtoon. OK — the list of named characters that cause hassle is currently this: Yu Han Sung (guy... honest, just believe us on this point), Ho (guy: no, seriously... don't let that hair or those clothes fool you!), Nya Nia (guy: even with that name!), Ran (nope, not a short-haired girl) and now we currently have Quaetro (undecided: as yet... we'll get back to you on that). Add to those the characters who, although you can tell their genders after a couple of glances, are still so painfully bishonen they can cause in-world issues: Baam (he can really fool), Agero, and... pop them in the right clothes, and you'd have problems with Hatsu and even Wangnan. Possibly even Leroro. Word of God is a good thing. Oh, and just to underline the point? SIU totally averts the trope with such testosterone-bleeding characters as Urek, Horyang (in his own, Gentle Giant kind of way) and Novick.
Because of the art-style and body structure, most characters in Attack on Titan are hard to tell which gender they are at first glance. Most notable offenders are Hanji and Nanaba, both officially women with strong features, but the author has said that people can interpret them however they like.
Ymir and Armin also have this problem but the anime made Ymir at least more feminine looking.
Ageha from Di[e]ce is another offender, wearing hotpants, mid-thigh-length stockings, a fitted jacket and a scarf which has a kinda flowery design on it. He also seems to have cat eyes and little pointy teeth, amping up the cuteness factor. Additionally, he is seen posing in a quite female or girly way, although the characters in the manga seem to recognize him as a boy immediately.
Shinobu from UQ Holder is probably female but just boyish enough that she could well be a boy with a girly face. In contrast to Kuroumaru, no deal at all is made over her gender, so we can't say for sure.
Kazuma from Summer Wars has a rather feminine voice and is androgynous enough that a lot of watchers have mistook him for a tomboyish girl.
Oyashiro-Sama from Higurashi no Naku Koro ni is used extremely mysteriously throught the series, and then the big twist comes in the 8th novel that Oyashiro is actually a girl. Deliberate trick on the viewer.
The Big Bad of Macross 7, Lord Geppelnitch, looks and sounds so completely feminine that most were convinced that the title "Lord" was a mistranslation. Several times the character is identified as male, but it isn't conclusively proven until the final episodes, where he appears without a shirt and the lack of breasts makes it clear. One fansubbing group felt the need to insert a note in the early episodes stating "No, really, that's a guy!" to pre-empt the confusion.
QT from Space Dandy, a robot with a female voice and mannerisms who takes on stereotypical female roles such as cooking and cleaning on Dandy's ship. Nearly everyone was convinced that QT was female. That is, until Dandy and Meow refer to him as a "he." The fact that they only start addressing him as male near the end of the first season didn't help.
Iori is a slender, long haired blond boy who's a part of the Sewing Club.
Not everyone realized Mataro was a boy until he's clearly shown naked and is referred to as Mako's brother. He strongly resembles his mother and sister, is prepubescent, and dresses like a tomboy. Many thought he was a Butch Lesbian.