Seiyuu Hiro Yuki looks, sounds, and acts like a 12-year-old girl despite being over 40 years old. Getting his sex right the first time is probably rarer than mistaking it. Also, another seiyuu, Kouki Miyata, says he's been frequently mistaken for a woman because of his high, effeminate voice.
Two female seiyuu examples: Romi Paku is often mistaken for a "New-Half" because of her "husky voice". Also, Wakako "Kujira" Matsumoto plays completely convincing older males (including Orochimaru, who also has his own entry in this page) because she sounds exactly like a guy. Whenever a female character has a suspiciously masculine voice, it's probably her.
Hyenas. The females are more dominant, aggressive, and "masculine" than the males. And the females have certain external characteristics that convinced Greek and medieval researchers that hyenas were hermaphroditic.
A lot of birds.
We humans are so used to the idea of human females being pretty that when one first encounters the world of birds, it can be rather jarring. You see, among many birds, the males tend to be pretty while the females are actually rather drab. This actually makes sense; if the female is the one caring for the young, you don't want her to be the one that's all eye-catching. Leave that to the male, who can draw a predator's eye away from the nest.
This is actually invoked in the narrative of Ed Wood's transvestite confession piece Glen or Glenda?.
With Phalaropes, it's the females who are the more brightly colored ones while the males take sole care of the young. This fact, can confuse those who are fully aware of the usual differences between the sexes in birds.
And with birds that color-wise look more or less the same regardless of gender — thinking of raptors here — it's usually the female that's bigger.
Then there are Chickens. Hens and Roosters are easy to tell apart when full grown but as chicks are indistinguishable. There's an actual job with training involved to be able to distinguish them as chicks.
This was invoked in Pet Shop of Horrors, where Count D invites a group of young society women and young society drag queens to tea at his shop. They watch a pair of feminine-looking dancers — who are in reality, male peacocks.
Parrots, on the other hand, look pretty much the same, and have to be DNA-tested to find out their sex. If your breeding pair aren't, it's often because they're both the same sex.
It's very difficult to tell male and female pigeons apart at first glance, unless the birds are courting each other. This isn't helped by the fact that they have many different color variations regardless of gender.
Ratites, the family of large flightless birds. Female ratites tend to be the dominant gender, being larger and more aggressive while males take care of young. Averted with ostriches, who are fairly easy to tell apart.
Human males used to dress up all pretty, and wear makeup and high heels, and this was perfectly masculine. It's only within the last 200 years that fashion forced men to be the dull drab dressers.
High heels were originally used by Persian horsemen to make it easier to stand up in the stirrups without the foot slipping through the stirrup (also why "cowboy boots" have high heels). They were in fact adopted by European aristocrats exactly because of how manly that was perceived to be. Obviously, though, high heels have become quite a bit higher after they became fashionable rather than functional.
Quite a few people thought Chris Crocker (of "LEAVE BRITNEY ALONE!!!" fame) was a girl. A few still do. Although to be fair, he did identify as male to female transgender until 2010.
Because his Author Avatar is genderless and one of the female characters carried the same name as his, Kagami Yoshimizu, the creator of Lucky Star, was wrongly thought as female by many — he lampshaded one when a male proposed to him at a Taiwanese convention, and now it seemed that even the American DVD release also confused his gender, calling him Ms. Yoshimizu...
Author Tracy Hickman has gotten so used to Viewer Gender Confusion that he sometimes introduces himself at fantasy conventions by saying "Tracy couldn't come, so she sent me".
Lady Gaga is often mistaken for a Drag Queen due to her androgynous facial structure and outlandish style. It also doesn't help that her haters started rumors claiming that she was either a hermaphrodite or transgender.
Christina Aguilera was accused of stealing Lady Gaga's style, and she even admits she didn't immediately know if Lady Gaga was a he or a she.
MAD once ran an article about all the plus-sides to the NBA mandating formal attire for public appearances. One of them was "You can now tell the difference between the NBA and WNBA players." The accompanying picture showed a confused autograph-seeker trying to choose between two tall, muscular, short-haired, earring-sporting b-ball players. However, you could just barely make out breasts on the one on the right.
As one story goes, the California police were looking for a man who had stolen a motorcycle. They found a man and bike matching the description and pulled him over... only for it to turn out to be 3rd Rock from the Sun actress Kristen Johnston, who is quite tall and broad-shouldered for a woman.
The new adverts for confused.com feature the logo as a woman while it was often assumed she was male. Apparently the logo was modelled after the founder of the website, Kate Armstrong, and was always meant to be a woman.
It seems fitting that confused.com would live up to its name in such a way, though.
Youtube guitarist makmakmakochan has pages and pages of comments debating their gender.
Australian singer/performer Courtney Act. Singer of the Top 30 (Australia) hit "Rub Me Wrong". Once made FHM's Top 100 Sexiest Women. Made the finals of the first season of Australian Idol, and even got a record deal out of it. He's a guy. Granted he is a drag queen, so it isn't too surprising he would create this kind of reaction. But even as a drag queen, Courtney is famous for looking extremely "fishy".
If you're over 30, you've probably heard the theme song to The NeverEnding Story, sung by Limahl. Even if you knew the (seemingly gender neutral, in fact seemingly made up) name of the singer, it's very likely you assumed he was a woman. Even watching the video doesn't help. He looks like a guy but sounds so much like a woman that you assume he's lip-synching. Seriously, listen to the song and try to find anything masculine about that voice.
According to Natalie Imbruglia, when she and Daniel Johns were romantically attached, a local paper ran a photo of the two holding hands in public, mistaking it for evidence of her being a lesbian. If you look at older pictures of Daniel Johns, this is sort of understandable, especially ones from when he was clean-shaven with long hair.
Brony musician, WoodenToaster and his OC Glaze. Many people were confused with the gender of Glaze. The reason for this was that Glaze has a female but without eyelashes and sporting a robotic male voice. The issue only got worse when the remix Love Me Cherilee came out, which showed a filly Glaze with eyelashes. WoodenToaster later explained in a video that the reason his OC is female is that he didn't like the "blocky" bases of the male ponies. It all became very clear to the ones who watched the video, but there are still many newcomers who don't know about the whole issue. Just go on any video having to do with the WoodenToaster and there will be at least twenty comments on the page asking if WoodenToaster is a girl or boy.
TV host Wendy Williams is often mistaken for a male-to-female transgender or drag queen because of her height (she's 5'11"), relatively manly features, and over the top wigs and make up. However, despite all this she is in fact not a man and in fact has a child and has had many miscarriages.
Ralphie, the live mascot for the University of Colorado Buffaloes football team, is a female bison. Every bison used to play Ralphie has been female, since the females only weigh 990-1100 pounds; the males weigh 1100-2100 pounds. The females are also more docile. With her masculine name, association with the football team, and the fact that she's a huge charging beast, most people assume she's male.