"That's 'No, Ma'am'"
"No, M... Wait, you're a woman?"
My first great triumph against impossible odds brought me a lot of attention. I think my favorite part of the whole affair was revealing to the waiting dignitaries who I am under my suit... The phrase of the day was "He's a whaaa?"
Must restore peace! Must escape! Peace in the universe is once again restored! HOLYCRAPIMAGIRL!!!
Whoa! Sorry about you suit…mam?! GUYS! We've been chased by a girl!
Wasn't expecting me to be a woman, were you?
Luigi: It's not fair, Link. The only people who flirt with me are other guys.
Link: Listen, if this is about that incident in '93, it was a really awkward time for me and I didn't —
Luigi: No. This bounty hunter guy tried to pick me up at the bar last night. Said he was only planetside for the evening and wanted to show me the time of my life. Couldn't even see his face under all that orange armor...
Link: Wait... Did this "guy" give you his name?
Luigi: Yeah. Began with a 'S'. Sam... or something.
Luigi: That's it! I told him I didn't swing that way...
Link: (facepalming) Oh, by Ganon's pork fetish, Luigi...
Luigi: What? What is it?
Link: Nothing. Not a gosh-darn thing. Say! You wanna come over and cry yourself to sleep on my couch for no reason whatsoever!?
But no living man am I! You look upon a woman. Éowyn I am, Éomund's daughter. You stand between me and my lord and kin. Begone, if you be not deathless! For living or dark undead, I will smite you, if you touch him.
— Éowyn, The Lord of the Rings
Akira: Brother, you're alive!
Daigo: I'm sorry, Akira. It must have been tough for a lady like yourself...
Edge: What? His voice... it sounds like...!
Gan: A lady like yourself?!
Akira: I'm sorry I've been deceiving you. I'm not the younger brother of the big boss... I'm his younger sister...
— Rival Schools: United By Fate
Oh no, please don't tell me I've been slaying hot chicks this entire time.
— Eddie Riggs, after one of the evil druids he was about to kill turned out to be an ally, Brütal Legend
Hold on, the Assassin is a chick? How did I miss that?
— The Assault, Monday Night Combat
Tony Stark: Good God, you're a woman! I honestly- I couldn't have called that. I mean, I would apologize, but isn't that what we're going for here? I thought of you as a soldier first.
Ramirez: I'm an airman.
Tony Stark: Well, you have actually excellent bone structure there. I'm kinda having a hard time not looking at you now. Is that weird?
— Iron Man
Gloomtail: WHAAAAATTT? You destroyed my precious younger sister!
Link: Hooktail WAS a girl!
Flurrie: Wait, you're Hooktail's brother?
Kirby: But what if "he" is a girl too…
Flurrie: No, I'm sure this guy's a…guy…
— Paper Mario X 2, Chapter 72: The Tail of Gloom and Its Doom
Chrom: Wait, you're...you're a woman?
"Marth": And quite the actress, too. Honestly, I'm surprised you didn't figure it out until just now.
Chuck: Are you a guy, or a lady? Because I never heard the name Samus before so I don't know if it's a male or female name, and your voice filter sounds kinda unisex so I have no way to tell.
(Samus Stares for a while)
Samus: Female. If that even matters.
Chuck: Thanks, sorry. Just wanna make sure I don't misgender anyone by accident.