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.
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.
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").
Moose (Margaret Rose) from Pepper Ann. 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.
You'd be suprised at how many viewers thought the title character was a boy.
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 confused for a little boy when the series was still new. It's possible that the creators eventually caught on to this, as one episode broke the trend of Limited Wardrobe to feature Mindy in a dress.
Later still, during a short which featured Elmyra Duff, Mindy was constantly, specifically referred to as a "cute little girl" by Elmyra. Almost as if to hammer it into the heads of the viewers.
It is actually a guy, although the high-pitched voice and eyelashes often leads to gender confusion, especially in foreign dubs where he is often voiced by a woman (while in English his voice was provided by a man, Mel Blanc). This is alluded to in an episode of Sylvester and Tweety Mysteries: 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."
Alluded to in an Around the World in 80 Days parody, where Tweety is seen coming out of a male (bird) restroom and says something to the tune of "See? I am a boy."
This has not been helped by Warner Bros. in the past few years, who noticing this, played it up — selling a line of Tweety Bird clothing for girls with him wearing bows and flowers in his head feathers.
Another source of confusion might be the title of the 1952 short Ain't She Tweet (although, in the context of the cartoon, "she" refers to the character of Granny).
His creator Bob Clampett explains that he's a boy during an interview when the host referred to Tweety as a girl. He has eyelashes and the high-pitched voice because he is supposed to be a baby bird.
Alluded to in The Looney Tunes Show where Sylvester is unsure if Tweety is a boy or girl, until he whispers it to him —- We don't hear it, but Sylvester's response: "Huh, I was wrong."
Speaking of Tiny Toons, god knows how many people have thought that Little Sneezer's a female. Given the Tertiary Sexual Characteristics (long eyelashes and high-pitched voice, same characteristics as Tweety mentioned above) deployed onto him, it's understandable.
And also from Warner Bros. we have the Road Runner, who is widely assumed to be female but is meant to be male.
Rorek, from Teen Titans (not the dragon) was mistaken commonly for a girl with the scarf over his face.
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.
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 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.
Cleared up in the recent episode, 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 by how Tuck's voice sounds that he's a boy.
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?"
Magenta is also a good version. Due to the fact Blue is female a lot of people thought, and still, think they were inverting the Pink Girl, Blue Boy, and gender roles, way of thinking. Thus Magenta is commonly thought to be male.
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.
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.
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.)
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.
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. Plus her name is rather gender vague and she does act like the boys.
Peppermint Patty, for those unfamiliar with the strip, due to her tomboyish appearance and being voiced by a boy in some Peanuts specials.
Translator Gender Confusion has also happened. In the English dub, she was male for around ten 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.
'Western Animation/'¡Mucha Lucha!'' has a few of these, though Snow Pea stands out. Snow Pea's young, cute and feminine, and wears a full-body costume. So it's hard to tell whether Snow Pea is a cute little boy or just 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.
Remember the baby from PB&J Otter? Butter was 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.
Many viewers have initially mistaken Rainbow Dash from My Little Pony: Friendship Is Magic as a boy. This also happened with Scootaloo. There's also a colt named Lickety-Split in Friendship Is Magic 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.
Many G1 ponies, especially the ones with deeper voices. Applejack and Gusty are prime examples.
Winnie-the-Pooh has Piglet: He is dressed in pink, has a squeaky voice (in some dubs, he's voiced by a woman), and has a bashful personality.
Rabbit is just as bad, if not worse. His voice sounds like it could be a high-pitched man, or an old deep-voiced woman.
Starlite from Rainbow Brite gets a lot of this due to his girly colour scheme and and the fact that his voice sounds like that of an old woman's.
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. While Tracy has a bob, s/he also has a very masculine body and wears boys' clothes. And there's also the fact that s/he has had different voice actors. It's Tracy who suggests 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 she 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.
This happened to Dora the Explorer in the early days. She lacked a lot of the Tertiary Sexual Characteristics associated with girls and had a rather gender neutral haircut. In the last few years this has been averted, as the series has become less and less unisex. A more straight forward example is Backpack. She doesn't have the most feminine voice, lacks any Tertiary Sexual Characteristics, and her coloring (light purple) is seen as a neutral color.
Edd (Double D) from Ed, Edd n Eddy wears a black sock on his head which is sometimes mistaken for his hair, which combined with his personality, has caused viewers to believe he's a girl. This happened in-universe in The Movie.