Rusty looked pretty androgynous when he first appeared, and they never said his gender (he was a "he" in the books). Then fans had a fit when the new writers called him a "she" in one episode. Writer Gender Confusion, anyone? Throw No Biological Sex (being locomotives, they shouldn't technically have eithernote Although a Viz parody completely subverted this) into the mix and you can take this anywhere you please.
Quite a few first-time viewers mistook Lumpy Space Princess for a male because of her manly voice.
B-Mo gets this the worst. The character has been considered male and female depending on the episode, but B-MO is actually an "it".
Jerry from Tom and Jerry, is a male, but he has a cutesy appearance, girlish eyelashes, and (when not punishing Tom), has a very sweet and affectionate personality. In some episodes he crossdresses and roleplays girls, and appears to enjoy doing so, and in a few others he actually kisses Tom. He generally acts very feminine, much like an annoying little sister, so it's clear to see why many people assume Jerry is a girl. In the 1993 film, he is given a slightly high-pitched male voice. Some assume that he is homosexual, but he is shown to fall deeply for female mice in some episodes, making him straight. Or perhaps a lesbian.
In early episodes of the My Little Pony 'n Friends and in the My Little Pony TV Specials it was never explicitly stated that Dream Valley was the ultimate Lady Land. Therefore, some fans assumed any deep-voiced Pony was male. Applejack and Skydancer tend to suffer this kind of confusion the most. Even decades later some people think Applejack is a stallion, though her FIM counterpart has helped the issue.
Many viewers have initially mistaken the tomboyish Rainbow Dash as a boy. This also happened with Scootaloo.
There's also a colt named Lickety-Split who has eyelashes, which are only displayed on the mares and fillies. This might have to do with the fact that Lickety-Split had been a filly in previous generations.
In "It Ain't Easy Being Breezies", pretty much all of the eponymous breezies look and sound very feminine. Including Seabreeze, who's male and looks like this. Note that he is voiced by the (very male) Brian Drummond...not that it's easy to tell as the pitch was cranked way up.
Some people still refuse to acknowledge that, despite being dubbed by a woman, Grumpy Bear is actually male. Latter installments of the franchise have him voiced by males, and usually somewhat deep-voiced ones also, so there's no mistaking his gender. More problematic are certain characters that flipped genders from one incarnation of the franchise to another, such as Funshine Bear.
Lotsa Heart Elephant, Cozy Heart Penguin, and Swift Heart Rabbit are this mainly due to the fact they're often presented as male or female depending on the media or episode. In The Care Bears: Adventure in Wonderland, Swift Heart Rabbit's uncle, Wonderland's White Rabbit, mentions (in passing) that Swift Heart is female. Many people, especially the Nostalgia Critic, were briefly shocked by this. Swift Heart's gender is the source of fan confusion, similar to Flaky from another cute-looking animal series. She's been referred to as male and female but the general consensus is she's a girl.
Garfield: Nermal is a male kitten, despite him given an obviously feminine voice in the cartoon. This might have been just to highlight Nermal's obnoxious cuteness before the mistake was caught, and wouldn't be really relevant except Nermal is still referred to as "he" in the show. It's just as bad in the comic, where he has long, thick eyelashes but is referred to as "he". They've seemed to fix this error in the most recent 3D movies by giving him a male voice actor (Jason Marsden) which makes him sound like a boy in the beginning stages of puberty. This is also averted in the first live action movie, where Nermal is clearly an adult with a very boyish voice. The Spanish dubs of the cartoon for Latin America actually went and decided to say Nermal was a girl, except for at least one episode.
Strika actually caused surprisingly little of this... Because her Beast Machines counterpart looked pretty much the same, and was also female, which meant that the gender confusion had already been gotten out of the way.
Transformers Prime: Soundwave has a few moments of this in his original appearance. He doesn't speak, simply repeats messages he's picked up—and the first he repeated was Arcee's, using Arcee's voice overlaid with his own computerized tones. This led some people to think he was female until he repeated another person's message.
One of the most oddly persistent examples is the German-Austrian-Japanese co-production Wickie und die starken Männer, known as Chiisana baikingu Bikke in Japan. Many, if not most, of the German-language viewers would swear that the title character is a little girl. This is slightly jarring, as he is regularly referred to as male — and the captain's son. There was even a scene where he was shown skinny-dipping, complete with a cutesy hint of male genitals. However, clothed, he certainly looks quite girlish. The impression is not only supported by the female voice actor, but also by a confusing bit in the title theme: one of the lines urges the character "Wickie, zieh fest die Segel an" ("Wicky, pull the sails tightly"). This was commonly misheard as "Wicky, sie fässt die Segel an" ("Wickie, she grabs the sails"). The English dub was even worse with this, using the title Vicky The Viking. The English dub of the CG reboot attempted to avoid this by using the title Vic the Viking, but characters still call the title character Vicky.
Moose (Margaret Rose). She's a girl, just very unfeminine. Even characters on the show have mistaken her for a boy due to her deep voice, refusal to wear dresses, and playing sports that are more commonly associated with boys like skateboarding, hockey, football, soccer, and softball. Also, Moose seems rather gender-neutral when it comes to names; anyway. Taken Up to Eleven in the Mexican Spanish dub, where Moose was voiced by a boy!
You'd be suprised at how many viewers thought the title character was a boy.
Some viewers also thought Milo was a girl.
The early 1990s animated Babar TV series featured a character called Pompadour, dressed in light orange, very effete and fussy, with a high-pitched voice and a tiny little bit of Ho Yay with Cornelius.
Mindy from Animaniacs was, despite her name, often assumed to be a boy when the series was new. It's possible that the creators eventually caught on to this, as one episode broke the trend of Limited Wardrobe by putting Mindy in a dress. Later still, during a short which featured Elmyra Duff, Mindy was constantly referred to as a "cute little girl" by Elmyra, almost as if to hammer it into the heads of the viewers.
Tweety Bird is actually male, but the high-pitched voice as well as the eyelashes often lead viewers to think that the character is female, especially in foreign dubs where he is often voiced by a woman. In the original versions of the shorts as well as the Hungarian and Hebrew dubs of The Looney Tunes Show, his voice was provided by men. His original voice actor was Mel Blanc. Another source of confusion might be the title of the 1952 short Ain't She Tweet. This has not been helped by Warner Bros. selling a line of Tweety Bird clothing for girls that depict him wearing bows and flowers in his head feathers. His creator, Bob Clampett, explained that Tweety was a boy during an interview when the host referred to the latter as a girl. According to Clampett, Tweety has eyelashes and a high-pitched voice because he is supposed to be a baby bird. This is lampshaded in an episode of Sylvester and Tweety Mysteries as well as an Around the World in 80 Days parody movie. In the former, when Tweety has to use the bathroom, he flies towards the female restroom before entering the male one and says, "I bet you all thought I was a girl, didn't you?". In the latter, Tweety comes out of a male (bird) restroom and says "See? I am a boy." The Looney Tunes Show also lampshades this confusion, where Sylvester is unsure if Tweety is a boy or girl, until someone whispers it to him. We don't hear it, but Sylvester's response is "Huh, I was wrong." In a later episode, Sylvester refers to Tweety with male-specific pronouns.
The Road Runner is widely assumed to be female, too, but is intended to be male. This is implied with some of Wile E.'s traps, which sometimes use female Roadrunner decoys.
Tiny Toon Adventures: God knows how many people have thought that Little Sneezer was female. Given the Tertiary Sexual Characteristics (long eyelashes and high-pitched voice, same characteristics as Tweety mentioned above) deployed onto him, it's understandable.
Rorek, from Teen Titans (not the dragon) was commonly assumed to be a girl due to the scarf over his face.
Avatar: The Last Airbender: Smellerbee was assumed to be male during her debut episode. However, since she was given a minor role, had an ambiguous voice actor (Nika Futterman, to be exact), hadn't hit puberty yet and was in a team of Lost Boys-style Freedom Fighters, you can't really blame the fans. Even the official site described her as being a boy. It wasn't until Season 2 that more fans realized she was female. Lampshaded, as Iroh refers to her as a "young man" when they meet, making her rather upset. Additionally, her French voice actor is a dude.
Another case of am ambiguous voice actor: Cooper from Ben 10. He had neck-length blonde hair and big blue eyes. At least in its Time Skipped sequel, Ben 10: Alien Force, Cooper got himself a haircut and a new voice actor. By the way, Cooper and Ben was voiced by a woman in the original.
A parent once wrote on the comments of the blog for Pocoyo that he/she (can't remember) was having a debate with his/her child over Sleepy Bird's Gender. The argument for Sleepy Bird being female is that she gives birth to a Baby Sleepy Bird in one episode, but the child argued that Sleepy Bird looks like a boy.
Linny the Guinea Pig from Wonder Pets. She wears a cape and baseball hat yet is female. She's the most gender neutral of the the gang. Linny is rarely ever referred to as female and always choose either masculine or unisex clothing. Plue she is supposed to be the age equivalent to a five year old, so you don't know if her voice is supposed to be feminine or it's just a five-year-old boy's voice. This was cleared up in the circus episode, where Linny's voice sounds (slightly, really slightly) more feminine, she wears a costume that vaguely resembles a dress, and is outright referred to as "she" by the ringmaster during her act. There have been a few episodes to showcase her gender, though most people seem to have been confused at first (her name sounds enough like "Lenny" to make that mistake, too). The irony here is that all 3 main voice actors are girls, though there's no mistaking that Tuck's voice sounds undoubtedly masculine.
The title character, Blue, is female, though many viewers believe her to be male. This is likely due in large part to the color blue being traditionally associated with boys. However, anyone who watches the show long enough will eventually learn Blue's gender, thanks to phrasing such as, "I'm looking for my puppy, Blue. Have you seen her?"
Due to the fact Blue is female a lot of people thought (and still think) the series was inverting Pink Girl, Blue Boy gender roles. Thus Magenta is commonly thought to be male. She's not.
Aside from Mr. Salt and Mrs. Pepper, you pretty much have to wait for pronouns to be spoken about any other object character. For instance, Shovel is male and Pail is female, but this is not obvious at all.
The character of Snail in the children's cartoon series certainly caused a lot of confusion for viewers. The character was a soft lavender color and had a voice that sounded female causing people to think Snail was a girl. (Actually, it didn't just sound female — it was female. Snail was voiced by Kristen Bone, Maggie of Maggie And The Ferocious Beast.) However, Snail is a boy. OK, technically snails are hermaphrodites... but since this is a kid's show...
Then there's Rabbit. Another character people tended to think was female, but was really male.
On the other side of the coin, there's Badger. Hmm... badgers are usually tough, fierce, and rather rowdy by nature... traits stereotypically associated with males. So, Badger must be a male, right? Wrong. Badger is a girl.
Likewise, Skunk is a girl but people have mistaken her for a guy.
Rocky of Rocky and Bullwinkle fame is identified as a male squirrel, but there is nothing masculine about him whatsoever. June Foray's voice for the character is distinctively female. To muddy the waters even further, in the Made-for-TV MovieBoris and Natasha, Rocky was made human... and became a woman.
Snooky Wookums the kitten from Krypto the Superdog is said to be male but, much like Nermal, has an obviously feminine voice and long eyelashes. To make it more confusing, in the episode where the dogs are turned into puppies, they call Snooky "nanny"; then later when Mechanikat turns into a kitten, he calls "him" "Poppa".
This is sometimes done by the production staff themselves. There are a lot of male background characters with effeminate names or appearances, which causes some confusion, such as boys standing in girls' bathroom lines. Emmanuel Maillard, a boy cursed with long hair, a gender-neutral(ish) name, and a necklace, has a girl's voice in one of his two speaking appearances. (His name was "Mike" in the other one.)
Poof from The Fairly OddParents, to the point where even his parents weren't sure if it was a boy or a girl when he was first born. Turned out Poof was a boy. It doesn't help that prior to the episode a game on the Nickelodeon site stated that Poof was a girl.
A large number of KaBlam! fans were shocked when they realized that June was female (she's got tomboy written all over her). Um, HELLO! Her name is JUNE!
Some people confuse Chowder for a girl since he wears a purple and pink shirt and is touchy-feely at times. The fact that his voice actor was pre-pubescent at the beginning of the series definitely doesn't help (his voice has dropped in later episodes, though).
Thanks to Grey DeLisle voicing Wubbzy in her brightest, most feminine voice, it's easy to forget that Wubbzy's a boy. Although Wubbzy does not eyelashes which helps somewhat.
Zero from INK: Invisible Network of Kids. She's a tomboy, she has a boyish voice and her eyelashes look more like face paint à la A Clockwork Orange.
Mitzi, the monkey, from Little Bear was female. This series is set pre-feminist revolution so it's justified; you wouldn't expect a girl to be so tomboyish in this setting, especially since the other girls are so feminine. Also, her name is rather gender vague and she does act like the boys.
In some Peanuts specials, Peppermint Patty, for those unfamiliar with the strip, suffers this due to her tomboyish appearance and being voiced by a boy.
Princess from Harvey Beaks is sometimes mistaken for a male because of her manly voice.
Translator Gender Confusion has also happened. In the English dub, she was male for around 10 episodes before the translators noticed something was a''miss''. Since then, she's been consistently referred to with the correct pronouns. However, considering the recent Gender Reveal, this could be accurate.
Some viewers have initially mistaken Stumpy for a girl due to his petite frame, high-pitched voice, jumpy nature and tendency to dress in drag. This also happens with Quack-Quack of all characters at times, likely because of his sheer non-information and the fact that he might look to some people as if he's wearing purple eyeshadow.
íMucha Lucha! has a few of these, though Snow Pea stands out. Snow Pea is a young, cute, and feminine character who wears a full-body costume. It's hard to tell whether Snow Pea is a cute little boy or a little girl. It doesn't help that all Snow Pea ever says is "Snow Pea". It has been hinted that Snow Pea is male, as he danced with a Distaff Counterpart once. If it helps, in the Spanish version he is called "Frijolito" wich actually means "snow pea", but in Spanish nouns are gender locked and frijolito is a male noun.
Anyone who watched PB&J Otter when it was on the air will remember the baby, but might not immediately remember her gender. Butter is a girl, though she's a baby and looks more like her brother than her sister, so it's very easy to mistake her for a boy. In the wedding episode, she wears a pink flower girl outfit like Jelly. The theme song also lists the family as "father, mother, sisters, and brother" but you have to listen closely to hear the plural sound. A few episodes did refer to Butter with female pronouns, but usually they just called her by name
Starlite gets a lot of this due to his rainbow colour scheme and and the fact that his voice sounds like that of an old woman's.
Twink is a cute Cartoon Creature with a squeaky voice. He is often mistaken for a girl.
Noodle from Gorillaz was often mistaken for a boy during Phase 1, but from Phase 2 onwards has definitely gotten more femenine. Bishōnen 2D sometimes gets the reverse, being mistaken as a woman by viewers from time to time.
While Alice is confirmed by the creators to be a woman, specifically male-to-female transsexual, there were fans confused and that thought she was instead transitioning from being female to male, that she was a male drag queen, or that she was simply a woman abusing steroids (or that her bulge was just a gag). Her origin in season 2 cleared up the matter, although it didn't help things for some of the fanbase.
Bruce, in contrast, didn't get as much debate as Alice, although there still was occasional confusion over whether he was meant to be a transgender man or a butch lesbian.
Although The Twins have always been referred to as men in the series (Alice's confusion over them aside), there were fans that had also mistaken them for women up until their shirtless scene in "Superbar". There are occasionally those that try to suggest that one of the two could be a woman, even though both refer to each other as "Brother" and have both been referred to as "boys" and "sons". A commentary track on the season 2 DVD had a guest who was even confused, asking the creators if the characters were meant to be two "ugly girls".
A one-shot character named "Peepers" in season 2 was meant to be a teenage boy, but due to being voiced by a woman, wound up having fans wonder if he was instead a teenage girl.
There's a minor character in Recess whose name is eventually revealed to be the gender-neutral Tracy, and the gender is completely up in the air. Despite having bobbed hair, Tracy also has a very masculine body and wears boys' clothes. And there's also the fact that Tracy has had different voice actors. It's Tracy who suggests that Spinelli was out getting more rocks to hit Randal with and it's clearly a male voice. In another episode where Gretchen tries to fail a test, Tracy encourages her and this time it's a female voice. In the "Picture Day" episode, the character is seen wearing a dress, so Tracy must be a girl.
Dr. Girlfriend in The Venture Bros., of course. Looks like a sexied-up Jackie Kennedy, talks like a stevedore. Plenty of in-house confusion - Pete White and Billy Quizboy ponder sex-change surgery at work, Brock suspects she's a guy, she tells a girl in a public restroom that she does belong there; she just smokes a lot.
Dora in the early days. She lacked a lot of the Tertiary Sexual Characteristics associated with girls and had a rather gender-neutral haircut. Starting in the mid 2000s this began being averted more, as she frequently appears in feminine attire.
A more straightforward example is Backpack. She doesn't have the most feminine voice, she lacks any Tertiary Sexual Characteristics, and her coloring (light purple) is seen as a neutral color. Eventually she was given a Girliness Upgrade in the sequel series.
Ed, Edd 'n' Eddy: Although it's been established that Edd (better known as "Double D") is in fact a boy, it's not hard for some people to mistake him for a girl—his black hat resembles long hair, he has higher-pitched compared to most of the other male characters, and he's somewhat effeminate compared to most of the other male characters. This also happens in-universe when Eddy's Brother refers to Edd as Eddy's girlfriend
Some first-time viewers of Grojband may mistake Laney to be a boy.
The title character of Maisy, an anthropomorphic mouse. She's a girl, but only speaks in a childlike gender-neutral babble and is often seen wearing clothes in colors that wouldn't be out-of-place on a boy, without any ribbons or other accessories that would peg her as a girl.
There are people who have originally thought Riley from The Boondocks was a tomboyish girl. What with his long hair, gender-neutral name, and voice (it's supposed to sound like a little boy but is somewhat feminine).
Many assumed Ruby was a boy because she is rather tomboyish and is an Official Couple with the more feminine Sapphire. Some people couldn't believe there would be a bona fide same-sex romance in a children's show, but Ruby is indeed a Butch Lesbian (of the discount variety due to Gems having No Biological Sex.)
Sadie's mother was thought to be her older brother or father until confirmed otherwise.
Pearl, especially in her earlier pilot design, often gets mistaken for a boy.
Jasper is very easy to confuse for a male; she's named after a stone that's also a masculine name, has a very raspy voice, is a giant, muscular war veteran with tiger stripes and was by far the most aggressive, cruel character at the time of her introduction.
This seems to be a common trend among all Yellow Diamond's servants. Though some of them possess reasonably feminine voices, all of them sport very gender neutral to rather masculine designs. Peridot only avoids confusion because she has a slight figure and a very high-pitched voice.
The Owl from The Owl & Co is supposed to be female, but aside from being pink she has no physical traits that make this clear.
Pickle the bunny from Endangered Species is female, but has no physical traits indicating this.
One thing about Bob's Burgers that puts some people off is the use of male voice actors for female leads Linda (wife/mother) and Tina (13-year-old daughter). In the original pilot, Tina actually WAS male, and named Daniel, with Dan Mintz using pretty much the same voice as he does for Tina.
For viewers that never read a book with is based on, Olive from Olive the Other Reindeer is female, but was mistaken for a boy due to her voice and has no physical traits.