In the Japanese version of Pokémon Black and White, there's a Hiker named Natsumi who you can take on a ride in the ferris wheel in the Summer if you play as a male character. He says some rather dodgy lines about how sweaty you both are and asks if you have a lover, but nothing very blatant. Then in Pokémon Black 2 and White 2, nearly any ambiguity was thrown out the window when they brought the character back. He talks about how he fell in love in the Summer 2 years ago (BW 2 takes place 2 years after BW) and how his heart was broken when he confessed his love. He doesn't use any gender specific pronouns about who he's talking about, but you can only talk to him as a girl in BW2, and he says he has "zero interest in your type." and he "Still considers you a friend even though you're a girl." The subtext is pretty obvious. His dialogue was completely changed in the English versions of BW, but oddly enough was left mostly unaltered in BW2.
Played for Drama in Assassins Creed with Abul Nuquod, who dresses very flamboyantly (both for the time and in general) and caresses his bodyguard's shoulder suggestively. However, he isn't played for laughs and is shown to be extremely resentful of the people and the social stigma he carries among them.
Assassin's Creed 2 has Leonardo da Vinci. His biography says he was probably gay and was convicted of sodomy (which is Truth in Television), but he is also very familiar with a brothel (which doesn't necessarily only have female employees) in Venice. The only thing for certain is that he really likes hugging Ezio. He became no longer ambiguous since the introduction of his assistant/lover, Salai (which might also be Truth in Television).
Rebecca went on a date with Shaun's girlfriend. That haircut isn't doing any favors either.
Shaun seems to find a lot of excuses to talk about gay issues in history, going on quite a bit about Leonardo's sexuality in the Animus database, and in Assassin's Creed 3 he writes an unreasonably long email to Desmond about homosexuality in 18th century America with no notable catalyst. He cares a lot about LGBT issues for a straight guy.
Maximillian Galactica, from the third case of Phoenix Wright: Ace Attorney: Justice For All wears pink, constantly says "fabulous", and calls Phoenix "sweetie". Although early in the case it's discovered that he proposed to a girl.
Miles Edgeworth from the same series, who reacts with nothing short of genuine repulsion when his coworker observes how popular he is with women, and is completely immune to the beautiful Dahlia, who almost every other male in the game instantly goes crazy for. (The Ho Yay Miles has with Phoenix doesn't help either.) Despite this, he's never shown to be interested in men, and nothing about his sexuality is mentioned at all.
Franziska von Karma, whose family Miles was adopted into, comes around the other way as Ambigiously Lesbian. She has short hair and a very short temper, and enjoys using her whip to deal pain to men who get in her way — but around women she can actually be very tender and caring.
Lana Skye is implied to have had an "intellectual attraction" to other women.
Adrian Andrews had a very close attachment to her mentor, Celeste Inpax, such that when she had committed suicide Adrian also attempted suicide. In general, she has a tendency to be dependent on other women. When she shows up again in 3-2, much more mentally stable, she then begins to show a dependency on Franziska. Made more obvious by a moment at the end of the third game, when Franziska takes Adrian away on holiday... supposedly "teaching her how to use a whip" in the process.
Aura Blackquill from Dual Destinies. It is heavily implied that she had feelings for Metis Cykes (also, they built two robots together in which both of them refer to Aura and Metis as 'Mama').
Aura: "Shut up, Simon! You know exactly how I felt about her! Her respect as a co-worker wasn't all that I wanted!"
Baten Kaitos has Geldoblame. In Origins he has an affinity for children's toys and cute things, a generally prissy demeanor (but he's an Imperial politician, so that's not a huge indicator) and keeps a scrapbook of another male character by his bed. He also takes that character's betrayal quite hard, almost as a spurned lover.
Banjo-Tooie has the glorious font of Getting Crap Past the Radar that is Jolly Roger. His mannerisms, his voice, his partner Merry Maggie looking like a guy in drags (and having one of the deepest voices in the game), then there's things like "Seaman's Surprise" and "Toad in the Hole". In Nuts and Bolts, Jolly now wears a purple robe, a belly shirt, and pink pants, screams like a girl if you hit him or attempt to run him over, and he paints Banjo's house pink at the end of the game! And he sometimes calls Banjo "Teddy Bear".
The Lazy animal archetype in Animal Crossing often fills out this role in games from Wild World onward, often when you least expect it. Some conversations between certain female character types also fill out a fair amount of weird Les Yay moments, including one such discussion where one animal comments that it would be cool if the other was made out of cake, so she could eat her thighs. And let's not even get into the Jock-type animal's strange obsession with spandex.
Joshua from The World Ends with You. His favorite color is pink; he loves rainbows, takes pictures of the main character's ass and calls him pet names; and as one announcer put it: "When he sees a hole, he knows how to fill it". Not to mention his constant girlish giggling.
This whole exchange right before Neku enters Shibuya River in Another Day:
Neku: I guess that's understandable. All right, I'll go check it out. Joshua: Whoa there, Black 'n' Blue. I'm going with. Neku: You? You seem the LAST person who'd want to go in there. Joshua: I could never live with myself if I let you go alone. Let's spend some quality time. Beat: Whoa ho ho! You guys are... Like, you ain't... Shiki: Blue and Pink!? Sure, they go.. but I didn't know they went LIKE THAT! Neku: Now look what you've done! Joshua: Hee hee... Shut up and walk, dear. Shiki: ... I'm going, too. If something happens, I want to see... I mean stop it!
And in the normal game, there's the priceless line:
Joshua: You watch my behind, and I'll watch yours.
He starts with a very high Brave stat, which lets him wear metrosexual shirts right off the bat and equip handbags with a bit of grinding. Also, the Frilly Parasol.
Fire Emblem Tellius has Ike. At the end of Radiant Dawn, he only gets a special ending with Soren or Ranulf if he gets an 'A' level support with either, in which he leaves the continent with said character. In the case of Soren, Soren vows to always be at Ike's side. Coupling this with how Ike never shows any type of strong affection toward females aside from his sister, Ike's sexuality is definitely in question.
Fire Emblem Awakening has revived the issue within the fandom: A secret character named Priam claims to be a sort of a descendant of Ike, but he cannot use Ike's trademark skill passed on through inheritance (Ike's father had it as well). It's ambiguous exactly what kind of descendant he is, since a wife is never mentioned, and his sister does get married (collateral descendance has been noted before in the series)... arguing about it causes many a Internet Backdraft.
Speaking of Soren, calling his sexuality questionable is frankly giving it too much credit.
Radiant Dawn also has Heather, though it can be debated whether it's actually ambiguous.
Ebisumaru from Ganbare Goemon is a glaring example of this trope, right down to the mincing gait and occasional crossdressing (not to mention his admiration of Goemon that borders on attraction). He's shown to be very interested in women, however, and to top it all off is the revelation of his daughter Mao at the end of Goemon's Great Adventure though no mention of it is made after that.
Mid-Boss and Master Big Star from the Disgaea series. This may be unintentional, due to differing Japanese stereotypes, but Master Big Star's character profile (for the Japanese version, anyways) did say one of his favorite things were "manly men".
Halfway through Disgaea 4, several of the characters start to suspect that Fenrich's obsessive loyalty towards Valvatorez isn't exactly platonic. After bluntly asking Fenrich if he swings that way and getting no denial on his part, Fuka just assumes he's gay from that point on.
Big the Cat from Sonic the Hedgehog. He often speaks with a lisp, he's been seen with a purse and parasol, and his two best friends, Amy and Cream, are girls.
The Sims has an odd take on this. In these games, NO characters are straight by default. The player can have his or her character enter a relationship with any other character, regardless of their genders, meaning the player decides their sexual preference when he does so.
In game, though, there are some pre-made characters who have been pointed out. Sims 2 gives us roommates Mitch Indie and Max Flexor, who are able to enter romantic reactions when first played, but won't automatically; Nervous Subject, who usually rejects flirts from women and may be autonomously attracted to men. Sims 3 gives up Gobias Koffi, said to prefer male company and unable to find the right woman; Alma Drill and Blaise Kindle, who are roommates who even have a pushed-together bed made of two beds; and most obviously, Viktor Valquist and Jesper Sandstrom, who, though called just best friends, are described as doing everything together, and the adopted daughter of one of them even has the surname of Valstrom
In Persona 4, Kanji's shadow and subsequent level is absolutely dipping with Flamboyant Gay and Ho Yay to the point of being both Anvilicious and offensively stereotypical. The game then builds an argument that Kanji may not be gay but is just questioning his sexual identity to the point that, by the time the game ends, the player isn't quite sure whether the guy is gay or not. If Extra Credits is to be believed, the developers at Atlus told Troy Baker, Kanji's voice actor, that he was gay in order to assist the way his character should act and speak. Persona 4 Arena confirms he does like Naoto, who is female.
'Punch-Out!! Wii gives Disco Kid this personality, just to round out the cast of stereotypes. He even calls himself FABULOUS.
Aera, from the second Swordcraft Story game of the Summon Night franchise. She behaves odd around girls, especially Lynn. Then again, Swordcraft Story is best known for its Lesbian options.
Razzy from the first game seems to develop a crush on the female protagonist, and even asks if she thinks that girls can marry other girls. This could just be admiration that she's confusing for love, but she only develops these feelings if you choose to play as a girl.
The Carecrow from Toonstruck behaves like a stereotypical gay man; he's very effeminate, he calls his crows his babies, and he's obsessed with fashion.
Zhang He from the Dynasty Warriors series is very flamboyant and focused on beauty, and these character traits only increase with each installment in the series. Compare: DW3, DW4, DW5, DW6. Yes, that is makeup he's wearing in that last one.
Balthier from Final Fantasy XII is definitely a candidate. Despite being a sky pirate that seems to woo most of the women in the game, the writers make him more irresistibly charming to them than actively pursuing any of the gender. His brightly colored bracelets and rings, concern for his clothes and appearance, and constant ambiguity in other such situations (such as his motives for helping the group) has made numerous fans suspect that something is up.
Fran does however state in the sequel that he did "something" to woo her and that his methods are different than the ones Tomaj tries in the same game. What he actually did and if they're actually a couple is left open however.
Debatable in Chrono Trigger. It's mentioned in one of the bonus endings that Cyrus plucks (or, in the Japanese version, pencils!) his eyebrows. On its own that just makes him vain, but then, consider the Ho Yay with Glenn...
Lucretia, Lelei and Cathari in Suikoden V. Lelei has been confirmed as lesbian and is hopelessly in love with Lucretia, who is implied to have been romantically involved with Cathari in the past. A few lines of dialogue (followed by a fade-to-black) between Lelei and Lucretia on the night before the big battle might easily be seen as a Relationship Reveal. It's a Suikoden game, of course, so these are only the most obvious examples.
Although his presence is brief, Belzeber from Star Ocean: Till the End of Time certainly gives off this vibe. He speaks in an eccentric tone, has very flamboyant mannerisms, and wears lipstick.
Flak and Shrapnel from Fallout 3. While Flak looks a lot like a stereotypical Leather Man and they share a bed, there's nothing explicitly stated about their sexual orientation. And this is a game with Discount Lesbians.
The bed-sharing may be an unintended side-effect of a script that requires them to be near each other. After all, this is known to cause problems with Shrapnel leaving Rivet City to wander the wastes if Flak has been enslaved.
Akira. Best friends with three girls and shows no interest in them, forcing yourself into the life of the main character that you just met, says double innuendo whenever near said main character, the best cook of the group, the only character in the game that didn't need Rui to tap his shoulder in order to remember Atsuki.
Hibiki. Shows no interest in women, hits on his best friend Ryo (who is oblivious to said flirting), gets jealous when Ryo hires a young co-worker at his bookstore, stalks Atsuki just to creep him out, rather feminine looking body, supporting Suzu's yaoi fantasy about him and Ryo being together.
Jak and Daxter: Razer in Jak X. Every other character says "Wham, bam, thank you ma'am!" as a victory line, even Ashelin. Razer, however...
Razer: Wham, bam, thank you... sir.
Some of his other lines are pretty suggestive as well.
Male Blood Elves in World of Warcraft, though more as a joke than anything. Not only is this a joke among the players but many of the male Blood Elves' /emotes, particularity /joke, dab in this trope. Then there's Martek's tale in the quest "The Day that Deathwing Came: What Really Happened." Ironically, male Blood Elves are larger and more muscular then the average real-life male.
Mega Man 8's Aqua Man, with his flamboyant posing, high-pitched voice and introduction.
The recurring villain from the flash RPG franchise Sonny, Baron Brixius, speaks in a voice that would likely produce a false positive on all but the most finely-calibrated of gaydars (with two of the three voice clips that play whenever he gets hit with an attack sounding more appropriate for wrestling than the armed combat generally seen in the games).
In Twilight Princess, the Lake Hylia clowns Fyer and especially Falbi gave off this vibe. Falbi wears a very small green belly shirt (it looks more like a sports bra with sleeves, poses with one hand on his hip, and the spoken dialog that plays when speaking to him is a high pitched "heeey". Fyer wears a pink shirt tied above his stomach (!) and has a broken heart pattern on his pants. Being minor characters, their dialog doesn't seem to point one way or the other.
Zelos from Tales of Symphonia. He's vain, feminine, wears pink, has long hair, and has several Ho Yay moments with The Hero (especially if his Relationship Values are maxed) including multiple instances of The Glomp mixed with Security Cling. The fact that he's The Casanova would set things straight, if not for the fact he's also very deceptive, which basically means it's impossible to discern his true orientation or lack thereof, and this has lead some people to believe his womanizing behavior is just another part of his facade.
Richard from Tales of Graces. Very elegant hand gestures, effeminate appearance, and quite possibly crushing on Asbel since they were kids. Not helped by the scene in which he gives Asbel his ring. Or the skit in which he pretends to be Cheria so Malik can practice flirting with her (It Makes Sense in Context). Or the fact that Pascal points out that they're "mwa-mwa hanky-panky all the time." Or the fact that he sounds extremely irritated by the idea of his many female suitors. Or the fact that he says things like "[Asbel] is such a kind man. It's the trait I love most in him." Or the fact that his entire motivation as a villain, though it's intensified by a little Demonic Possession, is the fact that he sees Asbel's rejection of his aid as a direct betrayal. And, as the ambiguous cherry on top, he gets a downloadable Zelos Wilder costume.
Dist the Reaper in Tales of the Abyss. A Sissy Villain to the max, he's completely obsessed with his former friend Jade, either to surpass him, force Jade to acknowledge him, or both, and desperately wants to return to the time when he and Jade went everywhere together as children. Refers to himself as "fabulous" and "gorgeous" (among many other adjectives), insists on being called "Dist the Rose", and generally acts like a jealous ex.
Hugh Bliss from the Telltale Sam and Max games. His voice sounds like that of a homosexual, he's obsessed with rainbows and "Prismatology", and according to Episode 205, one of the months on his Prismatology calender is called "Gaypril".
Seamus, the Vicount's son in Dragon Age II. He doesn't show any stereotypically camp traits, but if it had been the Viscount's daughter who kept running away with a Proud Warrior Race Guy to hide alone in the wilderness with the justification of "learn more about their respective cultures and enable peaceful cooperation between their people", nobody would have any doubt that she'd be really running away with her lover. This was confirmed by Word of Gay.
In Skies of Arcadia, there's something akin to this occurring with the pretty Ramirez in regards to how he feels for his captain and master, Galcian. In addition to having predominantly platonic relationships with every female he meets in-series - Fina, one of the heroes and his childhood friend, states that she viewed him like a brother and he treated her like that in turn - not long after his first mentor, Mendosa, betrays him, and he becomes far more cynical towards Arcadia's people, he clung to Galcian and his epic strive for global dominance. His loyalty is to the end, culminating in his crying and screaming for him when Galcian is killed, swearing to destroy all of Arcadia with him, and, in the Japanese version of the game, calling for him when he himself is killed (Ramirez is the game's Final Boss. Much like the game's infamously unresolved Love Triangle, the idea of Ramirez being in love with Galcian is pretty entrenched in the game's lore.
Taric was intentionally designed to invoke this trope, but there's also Diana, who Word of God has it might have had an unrequited crush on Leona, and might even still love her despite having become Leona's Arch-Enemy.
Pantheon is also suspect due to at least one instance of Word of God, along with his propensity for baking. However, most other creator opinions of him have the Spartan vying with Diana for Leona's affections.
There's also Vi and Caitlyn, though especially Vi. There's enough going on in their interactions that it would surprise nobody if Riot just came out and admitted they were a lesbian couple.
Tiny Tina of Borderlands 2. While her orientation is never explicitly stated, she tends to sexualize the female characters in her fantasies quite a bit, and one of the DLC sections makes it pretty clear that she has a Precocious Crush on Maya. And on the flipside, she pretty much never shows any sort of interest in any of the male characters.
Kenji Setoh from Katawa Shoujo. A He-Man Woman Hater with a penchant for pink bowling shirts, he started to fear and despise women (or better, feminists) after having sex with his then-girlfriend and believing that she sapped his life force. The writers put here and there some little innuendo in his conversations with male protagonist Hisao, and the background music from the bad ending with him and Hisao is the same one that appears during the sex scenes with the girls. Originally the writers intended Kenji to be Hisao's Gay Option, but this idea was scrapped because of time and other constraints.
Benimaru Nikkaido wears a cropped T-shirt, poses like a runway model, and has a soft, effeminate voice. His out-of-game biographies depict him as a promiscuous heterosexual, but the in-game portrayal has just gotten more ambiguous over time.