Doesn't help any that in the Spanish word for "tail" is feminine. And this hit the dub like a bolide hit Earth 65 million years ago.
A large portion of this is due to the urban legend that Tails was originally designed as a girl but was changed at the last minute. Said legend is prevalent both in Japan and the US, leading to double the confusion. Whether or not it's true has yet to be proven, but most informed fans believe it quite false.
If you research his design process, you find that the two-tailed fox design was always male, and that Sonic was always intended to have a sidekick the same gender as him.
It REALLY doesn't help that Tails' looks on the title card of Sonic the Hedgehog 2 looks really, really feminine.
Nor does it help that Tails is currently voiced by a girl.
Due to his dreadlocks, Knuckles is often mistaken for a girl.
Due to no voices, prepubescence, hair clips, and small graphics, some viewers originally thought Pat from Mega Man Star Force was a girl. The Japanese names for the character, Tsukasa and Hikaru, are both androgynous names at best. In addition, while "Tsukasa" is usually written in kanji for boys and hiragana for girls, Tsukasa Futaba Took a Third Option and writes his name in katakana. English names "Pat" and "Rey" attempt to keep this impression (or lack of it). You'd almost think it was deliberate...
Not to mention that the first time fans got a look at him was during the opening of the anime, where his hair was incorrectly colored blond. Needless to say, it didn't help.
Zero in Mega Man X comes equipped with long hair and, well, there's no other way to say it — booblights, causing some confusion until Mega Man X4 added his ridiculously manly voice, and ironically enough X's ridiculously girly voice.
Zero was always intended to be male. That bit of female!Zero art drawn as a joke just as R20 was about to be released in Japan didn't help matters.
Even worse was Harpuia in the Mega Man Zero series: a bishonen named after a always-female monster with a soprano voice in the first game. Later games pitched it down an octave or two.
And then there's Cubit Foxtar◊, which the Complete Works states as male (an oblique reference to kitsune, well, pretending to be women), and Polar Kamrous◊, who has a gruff voice to match her (this time the story stated it explicitly) build, from Zero 3 and Zero 2, respectively.
The first images of the "enemy Rockmen" in ZX Advent showed them in armored form, leading many to believe that Atlas (Model F) was a man and Tethys (Model L) was a girl; the names didn't help (Atlas was a Titan, Tethys a Titaness in mythology), and neither did the fact that they were boss-expies of Fefnir and Leviathan, also a man and woman, respectively. When the unarmored pictures and gameplay videos came out, Atlas had a visible bust and Tethys sounded like a boy, albeit young. They rapidly became known as "Trap" and "Reverse Trap" in the fanbase. The dub makes Tethys sound older and more boy-like, at least.
In the original ZX, if you never hear Hurricaunce's voice, you could think she's a guy.
But the worst is probably Lumine from Mega Man X8. Very female voice, long light-purple hair, thin feminine body, yet other characters always refer to him as a "he".
Similarly for Optic Sunflower, he has a very feminine voice, but is referred to as male in-game.
Eric Lecarde in the Japanese and European releases of Castlevania: Bloodlines is a bishonen-style pretty boy with a decidedly feminine outfit (a very short red tunic and white leather boots, and unlike the Belmonts, there's no armor or animal hides to toughen it up). The only tipoff you get is the conspicuous lack of breasts. The US version gives him a more masculine face to make it clearer that no, he's not supposed to be a chick.
Yoshi in Super Mario Bros. — "she" laid eggs for crying out loud. It really doesn't help that "Yoshi" is used to refer to either one individual or his entire species. Birdo is even worse though - a male crossdresser who spits eggs out of its... nose? Though given that the CREATORS have flipped on what gender the latter is...
Fan Wank often involves thinking of the "eggs" as more like poop, given the method of their production. Apparently Yoshis learned their combat techniques from monkeys.
Word of God says male Yoshis lay exploding eggs used for combat, females lay actual eggs, and the Yoshi usually seen with Mario and friends is in fact male.
Conflicting evidence comes from Japanese text from a Super Smash Bros.' trophy, claiming that Yoshis all have no gender.
Watt, the living spark in Paper Mario, has no obvious gender identifiers. The text usually uses feminine pronouns for Watt, but switches to male in at least one place.
It really does not help that Ruto actually calls Sheik a man at one point, or that Sheik's concept art looks pretty masculine...
Due to the artstyle making no body-built differences between male and female characters, quite a few players also thought Tetra, (who's basically Sheik's The Wind Waker counterpart, minus the mysteriousness) was a boy, right until Link's little sister Aryll exclaimed "How terrible! The girl fell into the forest!!". Tetra is the only one in the game who suffers from this, since she's the only female character who is wearing pants.
The guards found in the N64 Zelda games are a strange case: the official art◊ makes it easy to tell, but the actual in game model, due to graphical limitations, has a slender build with what appears to be a pronounced bustline, lipstick, and tight short pants.
There's a double example in Twilight Princess with the shaman Renado◊ and his daughter Luda◊. Because of his robe and her cowboy-like outfit, they could be easily seen as mother and son.
One Game Informer preview of Skyward Sword described Demon Lord Ghirahim as "a skinny, ghostly female".
Within hours of the reveal of the currently-untitled Zelda game for Wii U, there were a number of arguments as to whether or not Link is female in it. His Bishōnen attributes seemed to have been made even more prominent in this installment, with rather distinct eyelashes and a ponytail.
Others speculated that that's actually Zelda, as Aonuma stated that the character in question "wasn't necessarily Link", but later admitted he was joking.
Most protagonists all the way back to Cecil from the fourth game, who, upon armor removal, had long flowing hair and a tiara. Furthermore, he's got black lipstick in his concept art. Not to mention a fair few of the antagonists, until the tentacles and spikes and roots come out.
Speaking of Cecil, in Nintendo DS remake of his Final Fantasy IV game, he is given light blue armor and a blue hairband. He's still got the lipstick in the CGI, though. The voiceacting kinda helps, as well.
It's actually Lampshaded in Troia, when a drunken pub-goer mistakes him for a hostess.
Kuja from Final Fantasy IX, anyone? He's the dude back on the main page. He even has feminine hips and waist, which arguably falls into "cheating." The only thing keeping him from being a woman, visually, is that we can't see the boobs or junk.
This is lampshaded when he confronts Queen Brahne after she (expectedly) turns on him, and she says, "It's about time you showed your girly face here."
And lampshaded again in Dissidia: Final Fantasy where Penelo gives her descriptions of all the characters and claims that she dislikes Kuja since no boy should look prettier than she does.
The fact that his art by Yoshitaka Amano makes him look like he's wearing black lipstick and mascara (but then again, that's just part of his art style) doesn't help either. Surprisingly enough; Nomura actually made him look MORE masculine for Dissidia.
Amano's art for Kuja also has some stray sketch marks in the chest area that could definitely be mistaken for sideboob.
For that matter, at least one FFIX player mistook Zidane for a girl. But considering that they're brothers, maybe androgyny runs in the family.
All the Terran clones look identical to Zidane, even the girls, so no surprise there.
When Zidane specifically asked another of his kind whether they had males or females, they responded by saying that yes they do because asexual beings would lack the capacity to adapt to new environments.
Chances are that 75% of the people who have played Final Fantasy XII have mistaken Larsa for a girl. Alas he is a boy. A very girly boy. With very pretty shoes. A very twelve year-old boy.
It doesn't help that you can take him along on the hunt for Orthros, a creature that only appears with an all-female party. (Yes, you could also take the very manly Reddas with you much later, but Larsa's with you at the right level.)
Sorceress Adel from Final Fantasy VIII, obviously. Despite being a sorceress, the first reaction is generally "That's a man," due to her tattoos and overall masculine appearance. Not helping matters is the fact that Rinoa spends the entire battle attached to her torso, Adel siphoning HP out of her by thrusting her pelvis forward.
Ramza from Final Fantasy Tactics. Interestingly enough, Ramza's technically androgynous when it comes to the game's statistics system: he gets physical growth like a man and magical growth like a woman.
Many fans of Final Fantasy Tactics Advance thought that Marche looks like a female, with his near-feminine hair and shy personality. Luckily, when the bullies teased Marche for being "quiet like a little girl", Ritz's response made the fact that Marche is indeed a boy.
It doesn't help that Marche's hair actually looks just like Ramza's.
And after so many Bishōnen protagonists throughout the series, there were some that originally thought Lightning was yet another girly male protagonist!
And going farther back to Final Fantasy I, there was the White Mage. The most ambiguous of the six classes available, though class-changing to the white wizard implies that the class is intended to be male. 8-Bit Theater further popularized the depiction of the original white mage as female.
In the SaGa game Final Fantasy Legend III, a boss named Matreiya is referred to as "he" but take one look at the sprite and you'd bet that "he" looks like a "she"! S/he could easily pass for Cleopatra.
Partially justified in that some depictions of the Buddhist Maitreya could pass for female by Western standards.
Zelos in Tales of Symphonia upon first glance when you see him on the cover of the game... and sometimes second and third glances too. Once you meet him in the story and figure out he's a male-voiced Handsome Lech, you might be in shock for three more hours of gameplay. It probably also doesn't help that Zelos wears pink, and in the west, that's considered a feminine colour. (The Rapunzel Hair, too.)
Don't forget Corrine. Girly voice and name, cute and cuddly ball of fluff, multi-colored, Corrine's gotta be a girl, right? Nope. The little fox is a male. However, Corrine can also go into an even bigger gender debate when you take in two factors, the manga and the fact he becomes Verius. Verius is obviously male and outright proven so, especially when playing the sequel, and this is yet another factor that can confirm Corrine is male because he is Verius. However, the manga depicts Corrine differently where Verius is an entirely new entity sharing Corrine's likeness only in their life force and appearance. They also confirm Corrine is female and Verius is male. Oi.
Granted, a skit in the game states that Summon Spirits actually have no gender and only appear in either a male or female form for sake of convenience.
There's also Genis- he is referred to a he, but at first glance, a lot of fans thought Genis was a girl at first, especially given his hair, voice and his attachment to Lloyd.
Ion from Tales of the Abyss. Not only is he incredibly androgynous and has a very female-like voice, other people refuse to use pronouns to refer to him and instead say 'Ion'.
The Japanese version is slightly more difficult to confuse because his voice is more masculine and he uses a masculine pronoun to refer to himself... Mind you, there's a whole fetish around girls who do that, but most of those oddly act less feminine than Ion.
Jade gets a bit of this now and then, either from the especially feminine concept art or from the viewer only seeing a skit face, which shows the character from the shoulders up (he's quite pretty). The Gender-Blender Name doesn't help matters.
When gamers first saw the NA cover art of Tales of Vesperia, many assumed that the box was depicting the hero and heroine of the game. Nope. The feminine looking one with the long black silky hair is the Hero of the game. The Guy with the blond hair is the rival. The actual heroine of the game is on the back of the box.
This is lampshaded near the begining of the game. If you go into the bar and speak to a certain NPC, he mistakes Yuri for a girl and asks him to join him for some drinks.
And again, in Tales of Graces. You could be forgiven for thinking that the tall, beautiful blonde in the opening is a woman, especially since the video and the song are cut make it seem like she's Asbel's long-lost love interest or something. But no, he's a dude and his name is Richard. Some people actually interpret him as androgynous anyway, due to the genderless appearance of his Lambda form.
Fire Emblem has had a handful of examples such as Sephiran and Chainey, but the most amazing ur-example is Lucius, with his bright blue eyes, long, flowing blonde hair, and monk robes that made him look like he's wearing a form-fitting dress. Not a single person thought he was a man when they first saw his official art. His gentle, feminine, almost White Magician Girl personality really didn't help. Nor did his child-bearing hips. Or the fact that his main support conversation (and the only one with a double-ending for Lucius) is with another guy.
In the case of Lucius, it's even parodied in-game in one of his support conversations, where Serra mistakes him for a girl before he meekly corrects her.
Serra: B-But... to be a monk, don't you have to... be a... well... a guy? Lucius: Yes. Yes, you do.
Marth got this by Super Smash Bros. fans — partially due to the unusual first name, which is the male variant of Martha, and partly due to the pretty tiara he wears.
Fire Emblem Awakening brings another long-haired, blonde, pretty-boy monk (and likely a Call Back to Lucius) in Libra. Lampshaded to hell and back in the game, where he is routinely mistaken for a girl (and Sully, one of the most tomboyish Action Girls in the game, asks him for advice on how to look pretty).
Ironically, Sully can sometimes be mistaken for a male at first glance, especially as she has a very manly speech pattern and Boyish Short Hair.
Kingdom Hearts always had its share of gender confusion. If you started playing the series with Chain of Memories, it takes quite a while to get everyone's chromosomes right, no thanks to the confusing pixel anatomy and strange, quasi-corset Organization coats making EVERYONE have an hourglass figure.
Marluxia◊ was originally intended to be a woman. Due to "her" already slightly masculine appearance and the fact that this trope is par for the course for Square-Enix, they didn't actually have to change anything when they decided to make the switch. Amazingly enough, the Organization features only one lonely female◊ (but no shortage of estrogen).
When the promo images of Kingdom Hearts were first released, there was a debate on a Disney forum about whether Riku was a boy or girl.
Rhyme from The World Ends with You. Of course, she's a young girl, so unless others are talking about her with pronouns, there's no way to tell. Another example of going by the game cover; it's obvious in-game. Not making it any easier for those playing The Japanese version — turns out Rhyme is a Bokukko.
Many players have mistaken Terry from Banjo-Tooie as a female, since he gets mad because he thinks you've stolen his eggs, and no gender is immediately given, you'd think that he laid the eggs and is the female. But according to the game's instruction manual, as well as some random dialogue from Zombie Jingaling, Terry's wife has left him, and therefore he is very protective of the eggs.
Kanan from Soul Nomad & the World Eaters. In fact, you'll probably need to pick a certain path in the main campaign to find out "she" is really a "he".
Gunstar Red from Gunstar Super Heroes is a girl. It's due either by reading the manual or by her gender being expressed ONCE that you would know this.
This wiki even mentions that "Her name is Lotte." However; chances are most people didn't actually spot that name since the other characters have rather English sounding names (Jennifer, Laura, Bobby, Mary...)
The Game Overthinker summed Square-Enix's propensity for this rather nicely: "... Oh, wait, I think that's supposed to be a guy... Fucking Squaresoft!"
Ron Delite from Ace Attorney. He has all sorts of female characteristics, such as a pointy "egg" head shape, hair like that of Princess Leia and somewhat of a female face. Doesn't help that he's very kind, gentle and shy, and that his Mask☆De Masque costume is very husky.
Nick and Deanna from the Shining Force Gaiden series are some of the worst examples of this, ever. Just look them up, and be confused. It doesn't help that Deanna has a name that's a woman's name everywhere but in this game.
Khan from Shining Force III was a very effeminate holy man with a hat that covered everything on his head but his face. Also they only other character that used his weapons and fighting style was a girl.
Calintz, main character of the Magna Carta game (at least the one released in the West).
Secondary characters can invoke gender confusion in the player. Most notable of the examples is Milich from Suikoden 1 thanks to his extremely extravagant fashion tastes, the flower-themed palace, and noblewoman's laugh. Fortunately there is a way to know for sure if one is really that curious.
Some people had the same problem with Luc and Sasari in Suikoden II, looking at their portraits. The problem was rectified in the sequel, where both men had obviously male portraits and character models.
While almost immediately referred to as "Prince", preventing any confusion, the hero of Videogame/Suikoden V is downright girly. Only when you look at him at a certain angle (And while wearing certain clothes) does he even begin to look male. Most of the time, he looks pretty hot.
When Subala's character art was revealed, most of the fandom assumed that she was a boy due to her completely flat chest. Much to their surprise, she turned out to be a 16-year-old girl.
The Night Dancer in Final Fantasy Tactics A2 plays with this. He appears to be the first female bangaa ever in the series, but once you try to uphold the law in any mission he appears it quickly turns out that he is, in fact, a crossdresser (and no, the game doesn't generalize by race when it comes to unique characters, even though the law sounds like it).
The game also lampshades the confusion between two NPCs; a boy thinks the Night Dancer is an old man while another person mistakes the same character as a female. Even your own party makes a remark on the Night Dancer's gender and choice of clothes/make up, to which said character takes offense and claims he is "beautiful".
The Harvest Moon-iverse has Julius, who's... Well, just LOOK at him!◊ His favorite items are even things like jewelry and perfume! If it weren't for his name, it'd be next to impossible to tell that he's supposed to be a dude. And yes, you can marry him if you're playing as a girl. Sort of a shame, since there'd certainly be players who would marry him if he were in fact a woman. He doesn't even have the name to help him in Japan, where his name is Juli. The whole joke was that Candace/Kotomi was bullied by Juli as a child, and always thought he was a girl. Then she grows up, meets him again after so many years, and is shocked to discover that the girl who bullied her was a boy the whole time.
There's also Jamie from Harvest Moon: Magical Melody, who is always the opposite gender of your character, but they have the same sprite regardless (and hides this fact well by having Jamie wear a poncho the whole game).
In Fallout 3, many viewers were led to the impression that Fawkes was female, not because of any physical characteristics, but due to misunderstanding a laboratory log in game. After a bitter, lingering controversy, stoked by public comment from the voice actor who played Fawkes, the issue was finally resolved by Word Of God.
Closer observation of the in-game model of Fawkes would have resolved it even sooner: The tattered remnants of his vault suit has seam patterns consistent with the male version of the suit, not the female.
Thanks to voice acting being brought into the second game, NiGHTS from said titular game now speaks with a posh woman's accent, causing many people to think that they're a woman despite it being confirmed that Nights has no gender. NiGHTS' voice can be interpreted either as a woman or a young boy; it sounds equally like both.
Emilio Michaelov from Psychic Force, at least in the Japanese version, has a very feminime, timid voice, combined with his wings and even androgynious body shape and hair The US voice acting does remove this doubt.
Zhange He — Long hair, sometimes butterfly wings, pink and purple clothes, flowers EVERYWHERE. The only way you can tell his gender is by his voice.
Lu Xun — tiny, pretty, big eyes, slight body, looks like a prepubescent girl.
Samurai Warriors has Ranmaru Mori (who's even voiced by a woman) and is actually mistaken for a girl by Magoichi. The newest game brings forth Hanbei Takenaka, who is older than Ranmaru but still looks very feminine. The fact that he's seen disguised as a woman in his opening doesn't help either...
Uesugi Kenshin from Sengoku Basara, who is extremely pretty and graceful, has delicate features and is voiced by Romi Park. Takenaka Hanbe and Mori Motonari have similar problems, as not only are they pretty but they also have rather curvaceous hips.
Would you believe THIS, from Ty the Tasmanian Tiger had me thinking she was male? In all fairness, the voice acting sounds like a man trying to sound female, and bungling it, and from a decent distance, you can't make out the breasts. Also, Ty backs away in panic.
Arno from Summon Night 2: Swordcraft Story. Is it a boy, or a girl? Th characters never find out, Arno never goes by any sort of pronoun and him/herself a "child of the wind", and doesn't know their own gender...The only clue we ever get is one of the villains shouting "you neutered freak!" And it's not even a big clue at all. Seriously, you tell me.
Mao in Shadow Hearts: From The New World. That's a man's voice coming out of that cat, and yet it's supposedly a female. Since Mao's a cat without any form of Tertiary Sexual Characteristics, the confusion gets racked up. (She only refers to her gender a small handful of times, easily missed.)
Meet Bob. Bob is a kitty. Bob is purple. Bob loves to wear pretty dresses. Bob is a dude.
Likewise with Blanca, Gracie and Saharah are male... in Japan. A few villagers also get this trope.
You can also invoke the trope yourself by changing your hairstyle to one used by female player characters. Sadly, you can't wear a dress unless your character is female to begin with (except in New Leaf, which made dresses available for purchase in the clothing shop, including for male characters).
Wriggle Nightbug from Imperishable Night is sometimes mistaken for a boy, due to her being one of the few Touhou characters to wear pants instead of a dress.
This is not in effect for Mokou from the same game, despite also wearing something other than a dress— her name (lit. Scarlet Little Sister) gives it away.
The confusion strikes again, this time with Toramaru Shou from Undefined Fantastic Object, who does look overly masculine— tiger stripes and all.
And for a trip back in time to PC-98 days, we have the samurai Meira, whose battle challenge was mistaken by Reimu for a marriage proposition.
Teepo of Breath of Fire III. Long purple hair, acts like a tomboy, doesn't get referred to using a pronoun until literally the end of the game. Yes, he is supposed to be a boy. "He" also sounds distinctly female as a child.
Vivian, from Paper Mario: The Thousand-Year Door. In the Japanese release, she's transgender and her sisters treat her poorly because of it (arguably also out of jealously that a male could be so much more effeminate than they are). Like Birdo, this was changed for the US release, making her female from the start.
Also, Lopunny. There is a 50/50 chance that the Pokémon is a male/female, and it doesn't help that (s)he has a feminine appearance. It helps even less that most people say the name is a caricature of Playboy Bunnies.
A better example would be the Ralts line. While Ralts itself looks like a small child with a gender-neutral figure, the evolutions are ballerina based. However their "clothing" is feminine. As in "female tutu" and "dress" feminine. They gained a male counterpart the next gen, however you still have to go through the Kirlia stage and the males evolve into Gallade by a (somewhat rare) evolutionary stone; also, male Gardevoir still exist.
Even odder, Gardevoir has a very masculine name in Japan. Sirknight, which has both "Sir" and "knight" in it (while knights can be female, we typically think of male ones); though it may also refer to "come, Knight". To make it even worse, Mega Gardevoir appears to be wearing a ball dress.
N appears to be an ordinary Bishounen, but his character art uses Whitney's girly skipping pose, which has given some fans mixed signals.
This also could have occurred to the translation team for Ruby and Sapphire, who named a Team Magma admin with a slightly ambiguous-looking sprite the very feminine "Tabitha." Unfortunately, Tabitha is a man.
Braixen and Delphox are based on witches—Braixen, in particular, on the Cute Witch—but, like all other starters, has a 7-to-1 ratio of males to females.
Similarly, female Eevee are quite rare, yet a few of its evolutions, namely Vaporeon, Espeon and especially Sylveon look very feminine.
US players of the original Phantasy Star had a couple of examples. First there's the Bishounen Noah who is referred to by both gender pronouns thanks to the "Blind Idiot" Translation. Thankfully the re-release on the Gameboy Advance fixed it so he's always referred to as a male. Then there's the cat-like Myau, whose gender is never revealed in the Japanese version nor have I ever heard an Word of God on the topic.
Flea from Chrono Trigger. He's so girly that the entire party (in particular Marle) is shocked to learn he's a guy. Even his official artwork shows him as buxom. Of course, Flea's a shapeshifter who believes that "power is beauty", so he could be a man who looks like a woman, or a man who turned into a woman, or who the hell knows what.
Invoked in Baldur's Gate. An early sidequest has you hunting down a deranged necromancer serial killer, and when you track him down, he confuses you for his dead parent...of the opposite gender than you.
BioWare tends to avert this by giving the female characters either scanty clothing or large breasts. The only exceptions are Jack, who they make a point of not using gender-specific pronouns for her until you see her rising out of cryo, and Shale, who is a golem and doesn't give you any physiological clues as to her gender (in fact, even she is surprised by the Gender Reveal).
Brenda from Muscle March is officially female, but given that she's as ridiculously muscular as the other characters (with the exception of Rossi the polar bear), and the Macho Camp overtones of the rest of the game, it's easy to be confused.
Ranmaru from Sakura Taisen V. Always referred to with male pronouns... but only referred to with any pronouns starting in Chapter 6, when he's been appearing all game. With a high-pitched voice and very effeminate mannerisms. Given that he's most likely the same Ranmaru mentioned under the Art section, it's not too surprising.
Prince Pixel of Graffiti Kingdom. Granted, it's because he's a little boy and he's almost immediately called "Prince", but the fact that he has those huge eyelashes coupled with his female sidekick having a deeper voice than him makes it hard to believe that you're playing as a prince.
Kirby might very well be this, given that even the Japanese don't seem to know the character's gender. Most derivative translations default it as a dude, though.
Taranza was subject to this a lot when he was first introduced, particularly because of his hairstyle, though he's now generally accepted as male.
Ehl◊ from Solatorobo is a boy, to the surprise of many a gamer. The pink-ish fur and stockings don't exactly help with the issue. Subverted, Elh is actually a girl who acts like a boy, they find out her true gender thanks to a Shower Scene in Chapter 4.
Then there's Alman, who looks kinda female, uses somewhat affectionate/cutesey terms when talking, but is refered to using male pronouns.
Then the second playable Water Adept was revealed. A slender, MoeInegenue whose name in Japanese is phonetically identical to "Harmony". And an introductory storyline including an extended Shirtless Scene. The European official site cited the redubbed Amiti as a girl for a while, and we can't really blame them for being confused. It might run in the family; Amiti's biological father is heavily implied to be Alex, making Amiti and Rief second-cousins. Once you're looking for it, they even resemble each other.
Dark Souls: It's easy to mistake the androgynous Gwyndolin for a woman. It's how he was raised.
In universe example with Minogame from Hellsinker whose gender is described as impossible to tell from appearence. He/She is a Hermaphrodite.
In the original Rainbow Six trilogy, male and female operatives look identical in-game.
Dorrie from Super Mario 64 falls under this. Cutesy-looking plesiosaur with a feminine sounding name? Sounds like a girl, right? Nope, Dorrie's a boy. All official descriptions of the character refer Dorrie as a "he".
In the Etrian Odyssey series, you always have two male and two female character designs for each class. In several cases, at least one of the male designs could easily pass for a girl, while there are a few potential Bifauxnen. The Survivalists and Troubadours from I/II are prime examples, are are the Hoplite of III and the Swordsmen of IV. It's extremely likely this is far more deliberate than many other examples here, as well, since the player is encouraged to make up their own story in their head about their team in order to immerse themselves more fully in the game, so some portraits can be whatever the player wants them to be.
In Startopia, the Dahanese Sirens are the only race with human-like features, except for their dragonfly-like wings. They are also the only race to have visible sexual dimorphism. You have the larger broad-shouldered and bare-chested ones as well as the daintier swimsuit-wearing ones. Oh, and by the way, the first ones happen to be the females of the species, while the latter are the males. Of course, you won't know that without reading the manual.
Antasma from Mario & Luigi: Dream Team comes under this. Probably because he looks a lot like the (female) Cackletta from Mario & Luigi: Superstar Saga and has a name which could easily fit a female villain. And hey, he's only the second male antagonist in a series which before now has mostly had all female ones.
Freddi Fish was mistaken as being a male by several younger ones, mainly because she has a name more commonly heard in boys. She has a very feminine voice though, especially in relation to her much younger friend Luther. She's also identified as a female multiple times, including in the very opening scene of the fourth game. Some never caught on though.
Puyo Puyo character Oshare Bones is sometimes mistaken as a female, especially by Japanese fans as he uses feminine speech and pronouns in said language. Not only that, but he's also very Camp Gay, and seeing as he's a skeleton character with highly stylized anatomy, there really aren't any physical cues either. His English dubbed voice is easier to guess, however, since it sounds like a stereotypical gay male.
Earlier version of Best Fiends has Brittle the Housefly, who is hairy in her final form makes it hard to know if she is a girl. Later versions avert all of this.
The Plague Doctor from Darkest Dungeon wears the usual outfit of such an individual: many layers of very loose-fitting robes, a hood, and all-concealing beak mask. The outfit, combined with the dark, desaturated art style, leaves only the barest hint that there's a woman underneath all the layers. As such, despite descriptions referring to her as female, she's often mistaken for a man by players.