An Internet trope that was originally true, then an attitude in general, and finally a meme with only lingering traces left.
Back in the dim recesses of history, the Internet was predominately populated by young male nerds. While you might occasionally find a person with a genuine female identity in general anyone you met with a female username was actually male.
As the Internet grew, women became more common. Sadly, men pretending to be women became even more common as well; it soon became obvious that announcing you were female online (and thus a geek girl) was a surefire way to win lots of attention and accommodation from other users, even to the point of them being tricked into mailing gifts, cash, or performing other favors. This led to anyone claiming to be female to instantly be accosted with demands of "proof" (eventually codified as "Tits or GTFO").
Of course, proving you are female online is something of a conundrum, since any pictures or voice you present as proof can just as easily have come from a sister, girlfriend, or random site — which thanks to webcams was later solved by asking for a "timestamp", a picture with the current date, time, and a certain message (which could still be pictured with a woman who just happened to be near the actual poster, but it helps). Not proving it or refusing to share pictures means that you would be assumed to be male and only pretending to be female for the attention or (if in a game) free items.
This was a lot of fun for real women. Even a vague mention of a boyfriend or husband as an aside would get these righteous Internet College Males screeching in rage that you were an evil Attention Whore pretending to be female to get things. Girls naturally responded to this by pretending to be men just to avoid the verbal harassment and righteous screeching.
Part of the paranoia was justified on the early internet by both GIRLs and even some actual girls, taking advantage of the fact that the internet had a great number of socially awkward males who were easily duped into thinking this girl who was paying attention to them was seriously interested in dating them, if only they 'proved' their affection with gifts and special privileges. Nowadays, of course, attempting this usually elicits the aforementioned righteous screeching, deservedly so.
Nowadays, the lingering sentiment can only be seen as a meme in certain corners of the Internet and in people's assumption that anyone they meet online is male unless obviously otherwise. You can still see this to some degree in the gaming community, but that too is fading with time. With the advent of Voice over IP it's pretty obvious who's female (at least physically) as your voice will "give you away". It's still good to keep in mind that some people will not take your advice seriously and that sometimes the software distorts or changes the voice so that people will question your sex regardless.
There are, in fact, certain places where women now dominate the community, such as in Fanfiction sites, Soap Opera boards, Facebook, Second Life, LiveJournal and its clones (particularly in Journal Roleplay), Tumblr (the successor to LiveJournal for fandom blogging) or MySpace. As such, this is mainly a Discredited Trope these days.
See also Most Fanfic Writers Are Girls, Hackette, G.I.R.L. and Cross Player.
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Anime and Manga
Part of why so many people were in denial about Tsukasa in the .hack series. The series averts this in general, there's only two cases of a male pretending to be female.
In Cowboy Bebop, while the crew investigates the hacking of an old spy satellite, everybody assumes that Radical Edward, the mysterious computer genius who did it, is a male of indeterminate age, race, and apparently species. Turns out Ed is a thirteen year old girl (even when looking right at her, it took Faye a few minutes to tell).
Durarara!! Kanra=Izaya. And he's particularly girly online, leading to many fanworks of him crossdressing to get "in character." Also, it's suspected in the novels that Pure Water 100% is Aoba pretending to be a girl.
Subverted in that Setton (a basically androgynous user) turns out to be Celty, and Saika is a girl... well, originally a knife, but NOW a girl...
In Ultimate X-Men, Hank McCoy believes that he is spending his time chatting with a supermodel mutant named Naomi. It's actually the Blob, searching for info on Magneto's death. Cue things turning sour, when Hank accidentally lets it slip that Magneto's still alive and agrees to meet "Naomi", only to have Brotherhood telepaths rip their leader's location out of McCoy's head. Blob even says that everyone on the internet is either guys or guys pretending to be girls.
The Matrix: Neo is surprised that "Trinity" is a woman, he thought it was a guy. She replies that most guys assume she is a man, mocking the trope.
Earlier in the drafts, "Switch" was intended to be a female in the Matrix and a male in the real world.
Similarly, the protagonist of the movie Hackers is surprised that fellow hacker Acid Burn is female.
In Cube 2: Hypercube, the (female) protagonist is surprised to find out that the legendary hacker "Alex Trusk" is a girl, as the guy who originally told her about Alex assumed "he" must be male.
In a variant, the first victim in Surrogates is an overweight male who operated a sexy female surrogate.
Gamer: Kable's wife is an actress in that universe's version of Second Life, and she's "played by" a fat, sweaty pervert who likes to use her for cyber-sex.
In ReadyPlayerOneAech, the protagonist's best friend online, is actually a girl in real life but has a male avatar and persona. The Aech is pronounced as in H, and her name is Helen.
In OtherlandSam deliberately uses a male-persona online to avoid the annoying moths-to-a-flame effect.o
In Mike Nelson's Movie Megacheese, Mike interrupts his review of You've Got Mail to remind us that not only is Meg Ryan not chatting with us online, but that:
Michael J Nelson: "...no one who looks like Meg Ryan is chatting with you, either. No one named Meg is chatting with you... Guys, if there's a void in your life that you think is being filled online, keep in mind the person doing it has a beer gut and is named Dan, and remember that you're also filling a void in his life. Ladies... well, you don't need any advice, because you don't chat online."
Larry Nightingale::Me and the guys are trying to work out the other half. Sally Sparrow: When you say "you and the guys", you mean the internet, don't you? Larry Nightingale: How'd you know? Sally Sparrow: Spooky, isn't it?
Psych: "Poker? I Hardly Know Her!" - Shawn and Gus (well, mostly Shawn, but it was Gus' account) track down a guy by chatting him up online while pretending to be a girl, then asking to meet him. When they meet, the guy says that he should have known they weren't really a girl.
"All girls on the internet are guys. Except for you, who are actually two guys..."
On NCIS, McGee is very taken with a woman he's met in his online gaming program, the one where he's an elf lord. DiNozzo confides to Ziva that it's really him, messing with "Probie" and trying to teach him a lesson that women online aren't to be trusted. It's then subverted when McGee, tipped off thanks to Ziva, turns the prank around on Tony by Pulling The Thread and pretending to be getting a little too serious about the nonexistent woman, to Tony's great consternation.
This was kind of alluded to by Drew Carey on Whose Line Is It Anyway? once. His line went something like this: "Welcome back to Whose Line Is It Anyway, the show where everything's made up and the points don't matter. Yep, the points are something you'll never see, just like a real lesbian in a lesbian chat room."
Played straight in The Big Bang Theory: Howard and Bernadette had a breakup caused by him having online sex in a Warcraft Game with a female troll. Turns out the person playing the female elf was actually another male.
Way, way, averted in Person of Interest. The current Big Bad is a female black hat hacker who goes by the handle Root. (Bit of a Genius Bonus there: root access on Linux is the same as being an administrator on a Windows machine, which is what The Machine calls Harold.)
A variation is used in Foxtrot. Jason meets a fellow RPGer who he finds incredibly cool and talented. He is then horrified to learn that the player is a girl after she offhandedly comments that she has to get her bangs trimmed. Turns out it's Eileen Jacobson, who found out his username from his friend.
Peter was similarly afflicted, receiving an instant message from a bikini model who shared all his interests and wanted to chat; he assumed it was Jason pranking him and sent back a threat. It turns out it really is a bikini model with football posters on the wall, and she's calling the police as she reads Peter's message.
For a while, some users playing lady Night Elves in World of Warcraft got the idea of stripping off their armor and doing the /dance animation on top of the Ironforge mailbox for tips. Doing so was an immediate way to be branded a male gamer.
Sabrina Online mentions this quite early. When Sabrina logs onto a chatroom and the guys go nuts◊ when she announces she's really a girl. Granted it was 1997.
Arthur, King of Time and Space: In the contemporary arc, Arthur and Guinevere first meet when their characters on an MMORPG team up; Arthur initially doubts that Guinevere is really a girl, partly because she's playing a male character but mostly because she also claims that in real life she's not wearing any clothes. (She isn't lying: She's a nudist.)
Later, when their MMORPG team all get webcams, it turns out that Tristram is also female (which comes as a surprise to the reader, as well, since in the standard Arthurian tale Sir Tristram is male).
Though the big surprise around Tristram being female was actually because she had complained before about her uncle Mark's treatment of his wife Isolde (and she, like Guinevere, played a male character online) as if Isolde was just a trophy for Mark. The other characters thus assumed that Tristram was a guy expressing his frustrated crush on Isolde. Lancelot, as a reflexive homophobe (though he got better about it), definitely took the revelation the worst.
Frequently mocked (yet entirely subverted) in Enjuhneer.
The Slice of Life webcomic Friday y Innak explains that both the author Kkatman and her fursona, the eponymous Friday, intentionally chose masculine usernames because "I didn't want anyone knowing I was a girl. Lot of freaks on the internet." Innak, Friday's male room-mate, nervously replies "And now that you're one of them?"
Deliberately averted in The Guild, in which there about as many female gamers as there are male ones.
Inverted in Journal Roleplay. Female players outnumber the guys by a lot, and among many sources of drama is the community's tendency to view male players as "creepy."
In the Danny Phantom episode "Teacher of the Year," Tucker and Danny don't believe girls play online games. They're extremely surprised to find out the player Chaos, who routinely kicks their butts, is none other than their friend, Sam(antha), who then reveals that 'all' the best players of the game are girls, who know codes and secrets that none of the boys do.
Parodied in one episode of Duck Dodgers where he is playing an online fantasy game with a party. He initially assumes that the attractive blonde elf in the group is likely fat and unattractive in real life. Later he avoids getting a kiss from an extremely beautiful witch who he thinks the same of (he transformed her from a witch Hazel cameo after all), only for the audience to find out she looked exactly as attractive in real life.
On Futurama, Leela enters a chatroom and hears some nerds boasting about their "success" with women; She announces herself as female, only to have the nerds panic at her presence. Meanwhile, another "female" avatar speaks with an obvious male voice.
Of course, the chatroom they were in advertised itself as "Filthy," so...
In Undergrads, Gimpy (hacker alias G-Prime) catches another hacker hacking his computer. He automatically assumes this to me a male hacker until her name and logo comes up: She-Prime. He's initially freaked out.
Gimpy: Whoever did this is a man of skill! A man of cunning!
She Prime logo comes up.
Gimpy: A... A girl? A girl!?
Kyle from South Park plays as female fire mage in WOW in "Make Love Not Warcraft".
If you go by pictures and video, then pictures of girls probably outnumber pictures of guys - at least more than half.