It isn't at all unusual for siblings to be close to each other. Lots of siblings will hang out with each other and show some sibling affection towards each other. If someone else sees them and knows that they're related, they won't raise any eyebrows.
If they do not know that they're related, they might think the two are dating.
Oftentimes, the one who catches the two together is a lover or love interest of one of the siblings, and thus thinks that he/she is cheating or doesn't like her/him. Drama and/or hilarity ensues.
This trope is often used as a type of The Reveal where the audience does not know that the two siblings are related beforehand, and are led to believe that the person is cheating or likes someone else.
And, of course, this is most definitely Truth in Television.
This is somewhat the inverse of, but definitely comparable to, Like Brother and Sister. If the characters themselves don't know, it's Surprise Incest. If their resemblance to a romantic couple is particularly convincing, there may be Incest Subtext at work; if it's more convincing than the idea that they're brother and sister even to the audience who's already in on the secret, that's probably a Relationship Writing Fumble. Sometimes though, it happens to beExactly What It Looks Like.
Warning: Spoilers Ahead.
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Anime and Manga
True Tears, episode 3: Shinichiro sees Noe ride off on a motorcycle with another guy, who he assumes is her boyfriend. He finds out otherwise when he takes her to a basketball game and sees the same guy...
Shinichiro: (to his guy friend) That's him! That number four. He's Noe's... Noe: Brother! Shinichiro: ...brother. (pause) Brother?!
Used as the final reveal in the Di Gi Charat Winter Garden spinoff.
School Rumble plays with this one: After Harima sees Tenma and Karasuma together, he sulks in his room and ends up watching "Comet Wars", which mirrors the Star Wars use of this trope. Harima suddenly realizes he may have made this mistake and that Karasuma is Tenma's brother... but later realizes he was just fooling himself.
In Love Monster, Hiyo assumes that Kurou is flirting with Chiya, but in reality he just found out Chiya is his sister. Later on in the story, a squicky situation occurs where Chiya does actually try to pursue a relationship with Kurou, which is perfectly acceptable in monster society. Kurou is not interested however.
In Baka and Test Akihisa attempts to keep his sister who has just recently moved home hidden from his friends for fear of her embarrassing him. When they start getting suspicious and go to his house they believe that he has a secret live in girlfriend, much to Himeji's denial:
"Akihisa-kun, this bra is way too big for you. You need a smaller cup size."
In Weiß Kreuz, Omi and Sakura both assume that Aya-chan is their Aya's lover when they find out he's been taking care of a girl in a coma and using her name. The Assassin and White Shaman manga has a scene where Aya-chan forces her brother to leave his job early and buy her something to eat, and his coworker, implying that this has happened before, assumes she's his girlfriend.
The English dub of Sailor Moon. Serena and Mina mistake Amara for a man and assume 'he' is dating Michelle when they are actually cousins. Of course in the original Japanese they were a couple and the dub didn't edit out that many couple-y scenes between them.
Another variation in the R season. Serena/Usagi spots Darien/Mamoru giving another girl a lift and assumes he's found another girlfriend. The girl turns out not to be his sister but rather Andrew/Motoki's sister though the set-up remains the same. The sister has a boyfriend already anyway.
Macross 7 has a variation when Mylene visits Max Jenius on the bridge to talk to him. She's never been on the bridge before, and after they leave to talk in private, the Bridge Bunnies go into a frenzy over Max's new "girlfriend" who is so much younger than he is, some of them scandalized and others grudgingly admiring Max for attracting someone so young and beautiful. Then Exsedol cuts in and informs them all that Mylene is Max's daughter. (Mylene's somewhat risque manner of dress probably wasn't helping matters.)
Zig-zagged in K On High School when the other Pop Music Club members see Jun meet a guy. Three of them think he's her boyfriend, but Ui is unsure, having heard she has a brother, but they assume "boyfriend status confirmed" when she squeezes his arm. But then they learn after confronting them that he actually is her brother Atsushi.
Inverted in the Buffy the Vampire Slayer/Supernatural fanfic Runs in the Family, where Xander asks Sam and Dean if they're brothers, which Dean interrupts with "Dude! We are NOT gay!". Given the number of times they've been asked if they're together in the actual series, his reaction is understandable.
In Hero High many people think that Ben and Gwen are a couple. Though it doesn't help that the author enjoys adding the Ship Tease.
In the Buffy the Vampire Slayer/NCIS fanfic Little Brother, Xander Harris answers the door of his hotel room to find Gibbs and Tony waiting for him, wanting to ask if he knows anything about a murder in the same hotel the night before. When they hear their teammate Abby in the same room, sounding as if she's in pain (she had just woken up with a hangover), they flip out and nearly arrest Xander on the spot. It takes a few minutes until Abby herself sees this, and her angry reaction is "You cuffed my little brother?"
Six Brides For Two Sisters: Combined with Mistaken for Pedophile when Princess Cadence and Shining Armor recall how they first met: she saw him walking away with the filly she was foalsitting with promises of fun and candy, but since Twilight and Shining don't have any family resemblance (despite being siblings), she jumped to conclusions. Violently.
In The Dilemma, Ronny mistakes a man talking to his girlfriend as trying to feel her up but when he confronts them Ronny's girlfriend says that the man is her cousin. And gay.
The plot catalyst in Gray Matters. The brother-and-sister team both kiss the same woman, too.
Star Wars VI: Return of the Jedi: Han briefly fears that Leia's avowed love for Luke puts him out of the picture, and promises not to "get in the way." But having discovered Luke is her brother, Leia is able to reassure him. (Of course, considering how vigorously she kissed Luke in the preceding film, Han's doubts weren't exactly unreasonable.)
In Zathura, when Lisa finds out the astronaut she is attracted to throughout the movie is actually her little brother several years later, she cries, "OMG! And I wanted ta--" before an explosion cuts her sentence off. Needless to say, she was squicked and befuddled by it all.
Used in The Thomas Crown Affair (the one with Pierce Brosnan), where the young hot girl he's seen dancing with and was in his bedroom is actually his adopted daughter. He could've easily told Catherine this, but he triggered this trope because he wanted to test her.
Early on in With Six You Get Eggroll, Abby Mc Clure and Jake Iverson, both widowed with children, make a date for Friday night. Jake calls to cancel, citing his daughter's birthday, so Abby goes to a disco with her sister and brother-in-law, where they see Jake dancing with a pretty young girl. Abby gives Jake the cold shoulder, but later learns that the pretty young girl is actually Jake's daughter when she sees her at her son's high school graduation.
In Letters to Juliet, this causes Sophie some grief when Charlie introduces her to Patricia, his ex-girlfriend. It turns out that Patricia was also the name of his cousin whom he was really introducing.
This trope is a great staple of the literature of the 19th Century, as it allows the conflict and drama entailed by (usually) a woman flouting social norms, while still keeping the heroine innocent. Jerome K Jerome takes a moment to complain about theatre plays relying on this trope far too much.
In Susan Calvin's introductory story, a lying robot persuades her that she's made this mistake about a love interest.
The Tenant of Wildfell Hall by Anne Brontë. The hero, Gilbert, becomes suspicious of Helen's close relationship with her landlord Mr. Lawrence, and knocks the poor man unconscious in a fit of jealous rage. Of course, Mr Lawrence is Helen's brother. (As she's living under a false identity to evade pursuit by her alcoholic husband, they have kept their relationship secret, but their clandestine meetings have fueled gossip in the village.)
In Blood Rites, between Thomas Raith and Harry Dresden, made worse by the fact that 1) they cannot openly reveal their true relationship, and 2) they were sharing the latter's small apartment for an extended period after Blood Rites. And 3) Thomas was deliberately pretending to be gay in his public life for completely unrelated reasons. Considering all this together, we should have seen it coming from the paragraph after that "Wizard of Oz" poster on Thomas' wall.
In Dead Beat, Waldo Butters (after meeting Thomas for the first time), is hurt that Harry hasn't previously come out to him. Thomas deliberately kisses Harry on the head and pours him tea after listening to Harry's subsequent startled denial.
In White Night, this is deliberately exploited to explain to building security why Harry has a key to Thomas' apartment and has turned up there in the latter's absence.
In The Count of Monte Cristo, Debray assumes Albert and Mercedes are having an affair after running into them at a seedy inn. The fact that Mercedes is veiled partly excuses the fact that he mistook his friend's mother for his mistress.
There's also an earlier scene where the Count sees a painting of a younger Mercedes in peasant dress and makes a comment about her being Albert's mistress. While he's only pretending to make that mistake and actually knows who Mercedes is, it's kind of weird that Albert keeps a picture that his mother had made for his father as a romantic gesture note Fernand didn't want it because it reminded him of his past/his betrayal of Dantes, and explains why he has such a massive Oedipus Complex in Gankutsuou.
North and South by Elizabeth Gaskell. Similarly, the heroine Margaret Hale's reputation is damaged in the hero's eyes after she is observed saying an intimate goodbye to a young man at a train station, and then lies about it. She cannot reveal that the young man was her brother as he is a wanted man.
In Bag of Bones by Stephen King, Mike learns that before his wife Jo's death she had been seen hanging around TR-90 with a man and being openly affectionate with him. He naturally assumes that his wife had been having an affair until he confesses his fears to Jo's brother - who is very quick to explain the truth.
The Wouldbegoods: This trope is cheerfully lampshaded. The child protagonists are reluctantly trying to reunite their adult friend with his long-lost sweetheart even though they fear this will mean he has less time for them. So they are pleased, rather than scandalised, to find the girl embracing another man. But their relief is short-lived and the narrator reflects, "He might have known it was her brother, because in rotten grown-up books if a girl kisses a man in a shrubbery that is not the man you think she's in love with; it always turns out to be a brother."
Sue Townsend's Adrian Mole, when aged fifteen, is allowed to witness the birth of his sister because the nurses assume he's actually the baby's father.
In one of Kathy Reichs' Temperance Brennan novels, several other characters mention seeing her boyfriend Ryan going out with a young woman. Its eventually revealed that she is his recently discovered daughter.
Averted in Northanger Abbey when the heroine sees her Love Interest with another woman on his arm and immediately comes to the correct conclusion that it's his sister, thereby depriving herself of a perfect opportunity for needless drama, as the narrator tells us.
One of the stories of The Berenstain Bears had classmate Queenie McBear put an unapproved article in the school newspaper about a teacher being seen having coffee with a pretty teenager, causing a fuss and an inquiry for unprofessional behavior. Turns out his teenage niece had been visiting him.
Inverted in Little Women. A suitor of Jo's calls at the house and is informed that "Ms March and her husband live next door". He assumes Jo has married someone else but Jo hurries down the road after him to explain that it's her sister who is married.
Live Action TV
In Black Books, Fran gets annoyed when she sees her boyfriend having coffee with another girl, that turns out to be his sister. Partially lampshade hung when Bernard guesses, correctly, that that was the case before Fran actually reveals that to him.
Used secondhand in Bones where Booth and Brennan saw Sweets' girlfriend in a wedding dress hugging another man, and after some disagreement Brennan told him, made worse by her directly informing him that his girlfriend was engaged to someone else. Luckily for them when he confronted her about it she explained what had actually been happening (her trying on a wedding dress for her cousin, who's the same size), and they... reconciled.
Friends: Rachel is shocked that her boyfriend seems to be cheating on her, only to find that the woman he was hugging was his sister. Then Rachel goes to their apartment and finds them tickling each other, eating whipped cream from each others' laps and, ultimately, sharing a bath together. Her boyfriend insists that it's all innocent and just proof that they're close enough as siblings to behave as they did as kids, Rachel thinks it's too weird, and leaves him.
Sibling Ross and Monica Geller, who in spite of never having on-screen moments that may confuse people, are shocked to learn in the tenth season that they had actually kissed while teenagers (in fact, Ross was Monica's first kiss ever).
Two and a Half Men features brothers Charlie and Alan acting a lot like a married gay couple, which is a running gag in several episodes. For instance, in one occasion Alan's out camping, Charlie's asked by a friend about "the wife and kid".
In Keeping Up Appearances, when Hyacinth sees Emmett, her neighbour Elizabeth's brother, for the first time, she believes her to be cheating on her absent husband. Although, it isn't because she sees him with her, it's because she sees him at her house, dressed only in a towel.
One episode of Power Rangers Lightspeed Rescue had Ms. Fairweather call a famous scientist named Clark to help her build some armour for the Rangers. They get along exceedingly well, and Clark is even given the honor of revealing her first name (Angela) to the audience. Meanwhile, Joel, who was in love with her, is seen getting very, very jealous, and even tries to sabotage one of their 'dates.' Finally, they get attacked by a Monster of the Week and, seeing Ms. Fairweather's obvious distress when Clark is still in danger, Joel decides he wants his beloved to be happy and saves his rival. Later, he gamely tells her how glad he is for her and that she deserves the best boyfriend ever. Naturally, this is when he is treated both to the reveal that he was her brother all along and to a kiss on the cheek. In a No Hugging, No Kissing series, even. Joel and Ms. Fairweather have the distinction of being the first PR Official Couple to get married.
Remington Steele: Steele once saw Laura talking with her brother-in-law and thought she was having an affair. It didn't really help that the brother-in-law kept kissing her on the cheek...
The brothers in Supernatural are frequently mistaken for a gay couple. This apparently does not stop being funny, ever. In this Crapsack World you take laughs when they come.
In the last season of Veronica Mars, Mac's newly introduced animal-rights-activist love interest turned out to have a female housemate, who triggered this trope.
Variant: An earlier episode had Geraldine nearly call off Alice and Hugo's wedding when she thought that David Horton, Hugo's father, was also Alice's dad via a one-night stand. Turned out the David in question was a relative of the show's David who had the same name.
Firefly: When the box containing River is opened, Mal says scornfully, something to the effect of "Is she your lover, because you do seem awfully worried." Specifically, he accuses Simon of human trafficking, then sarcastically says something like "Oh, I'm sorry. Is this one for you, is it true love?"
Used in The Fresh Prince of Bel-Air, in which Hilary and Carlton were each flirting (separately) with an online admirer. They went to the restaurant to meet their date. . .only to discover that it was each other.
Full House had an episode where Kimmy and Stephanie see DJ's boyfriend Steve with a woman who turns out to be his cousin.
In Desperate Housewives, Lynette pretends to be a teenage girl in a chatroom so she can have real conversations again with her son. Unfortunately, he gets a crush on her online persona and she has to let him down easy with a goodbye message...which she signs "Love, Mom."
Inverted in Malcolm in the Middle, where Reese walks in on his wife cheating on him and she tries to pass off her lover as her brother. Having turned into an Extreme Doormat by then, Reese buys it, and even continues to call him her brother when he later walks in on the two of them in bed.
An episode of Frasier had Niles shopping for an engagement ring, accompanied by Frasier and Martin. Everyone in the store assumed the brothers were getting engaged to each other and applauded, much to their father's dismay. Seeing as how they do everything together, including going to their high school prom, this has probably happened to them a lot.
How I Met Your Mother featured an extended flashback to Barney's first love. His girlfriend was supposed to go into the Peace Corps with him, but bailed at the last moment with the excuse "My father won't let me go." Barney goes to give the father a piece of his mind, and walks in on them kissing.
In an episode of Drake & Josh, Josh sees his ex-girlfriend Mindy hanging out with and being affectionate with what he assumes is a new boyfriend. Of course, he turns out to be her cousin. When Josh asks why Mindy didn't say so in the first place, she says that Josh wasn't letting her explain.
In one episode of Brazilian TV show A Grande Família (The Big Family), Isabel "Bebel" Carrara assumed her husband was having an affair. Turns out he was just meeting his long-lost sister.
Tracy Barlow invokes this in Coronation Street when her brother Peter is upstairs as her abusive boyfriend Charlie arrives home. Tracy pretends that she has brought a guy home but quickly shouts that it's her brother when Charlie rushes upstairs. Unfortunately he finds Peter shirtless (he'd just had a shower) and beats him up.
In True Blood, Violet Mazurski assumed Jason Stackhouse was cheating on her when she saw him with Sookie until he told her Sookie is his sister. Violet's response, "Then you're my sister too!" and then kissing her on the lips.
A darker variation occurs in The Tailor Shop on Enbizaka from Vocaloid. The tailor sees her lover with three different girls on different occasions, much to her annoyance. It turns out that her "lover" never even met her before and the three girls were his wife and two daughters. Who the tailor all murdered in a jealous rage, including the man as well.
This is the plot of "After the Ball."
The Gospel duo of Bebe and CeCe Winans could come across this way to people who don't know they're siblings. They have a very close relationship and mixed-sex duos who are married are more common than siblings.
In the Gilbert and Sullivan operetta Iolanthe, the twenty-five-year-old protagonist Strephon is caught embracing a young woman (actually his mother). Since his mother is an immortal fairy and, as such, appears to be about seventeen, Strephon's fiancee is understandably skeptical and promptly breaks off their engagement.
Badly Drawn Kitties used this once. Lucy overheard Lydia's boyfriend Dirk and another woman declaring their love for each other, and assumed because of the woman's foreign accent that she wasn't related to Dirk. It turned out she was in fact his sister and she'd just been studying in England, and so had picked up the accent;
"And you're not sleeping with Dirk?"
"Uh, no. I said England, not Georgia."
Given that Lucy had had a "unique relationship" with her own brother, that wasn't actually an unreasonable assumption on her part. But in the original continuity, Lucy and her brother were originally from Georgia (the US state), so there may have been some Lampshade Hanging in the rebuttal.
Ménage à 3: Zii makes this mistake, then, panicked, tries to cover herself under it. (Zii is new at monogamy.) Her boyfriend is sweetly understanding.
Done in Too Much Information, when the two main girls of Ace's Unwanted Harem sees him driving a pretty, blond girl around in his sports-car... it's actually his cousin, whom he's giving a ride to an abortion-clinic so her parents won't find out.
In Kevin & Kell, a well-intentioned Kell sets her friend Aby Eyeshine up on a blind date with Rudy's hunting coach, Sam Pardus. Sam and Aby, in accordance with the trope, are brother and sister, but Kell didn't know that (all parties found the whole thing amusing). Sam being disguised as a leopard (while Aby is a house cat) and changing his last name in order to coach the Caliban Academy team despite being domesticated makes it that much harder for Kell to find out.
Captain Planet: In the episode "Missing Linka", a disguised Wheeler helps saves the life of Linka in her hometown in Russia. He believes Mishka is Linka's old boyfriend, but in the end, like it turns Mishka is Linka's older brother.
In Garfield and Friends, "Wedding Bell Blues", Jon's cousin Marion, in town for her upcoming wedding, comes over to visit. She says the groom-to-be is named Harold John. When she points out the coincidence, and that, as a result of the wedding, she'll be "Marion John" (say it out loud), Garfield interprets the remark as the woman being Jon's secret fiancee and spends the episode worrying that Jon won't pay as much attention to him after the wedding, but more that their children will play harmful games with him. At the end, he discovers what the viewer knew from the beginning: that the woman is Jon's cousin and she is marrying a man whose last name is John. His attempt to disrupt the wedding is fortunately taken in good humor and the wedding continues.
My Little Pony Tales: In the episode "Sister of the Bride", Clover and her friends see Cheval (the groom) with another girl and accuse him of cheating on Meadowlark (the bride). Eventually, Cheval explains that the girl is his sister.
The Simpsons: Lisa has a crush on an older boy, then hears him having a phone conversation with a girl, saying that he can't wait for her to get there. When the girl arrives Lisa purposefully gets her lost in the woods, only to find out that her rival is, of course, the boy's sister. (The boy, naturally, does not take this well.)
In the Phineas and Ferb episode "De Plane! De Plane!", Candace becomes jealous when she finds her crush, Jeremy, "flirting" with a prettier girl who turns out to be his cousin.
In an episode of The Weekenders, Tish requests that she just hang out by herself for the upcoming weekend...and the others spy on her, suspecting ulterior motives, and see her hanging out with a boy her age all over town. When they confront her about her 'new friend,' she reveals first of all that it's her cousin Doug, and secondly, she had planned on being by herself that weekend, but Doug's visit was last-minute and her parents asked her to show him around.
In an episode of X-Men: Evolution Mystique brings Wanda into the Brotherhood house and Pietro has a huge Oh Crap moment when he sees her. During her rampage around the house, Fred asks if she's an ex-girlfriend. Pietro responds "worse - she's my sister".
On Regular Show, Mordecai sees a man bending down in front of his crush Margaret, and overhears her saying "Yes dude, yes!" Thinking the man was her fiancee, Mordecai spends the episode in a depression. At the end we find out that the guy was her cousin, John, and the reason he was bending in front of her was because he was showing her his prosthetic leg.
In the last episode of Road Rovers, Hunter tracked down and went to visit a female dog from his past. Everybody, particularly Colleen, thought she was an old girlfriend. Turns out, she was his mother.
On Family Guy Brian is trying to build a friendship with Quagmire, who hates him. At one point he sees a disheveled woman in Quagmire's doorway and, knowing the kind of guy that Quagmire is, starts making jokes about them having apparently just had sex. Quagmire angrily informs him that that's his sister who came over to escape her abusive boyfriend.
Quagmire: I also have a deaf brother! Want to make fun of him, too?!
Ex-England football team coach Sven Goran Eriksson was snapped with a young blonde. the Daily Star "newspaper" ran a front page story, basically emphasising the fact that she was young, blonde and worth a few "Phwoar!"s. The girl turned out to be his daughter.
The "Kiss Cam" at any sporting event usually makes this mistake every once in a while.
Including one relatively well-known picture where a man actually holds up a sign with an arrow pointing to the woman he was sitting with and text saying "My Sister" when the kiss cam focused on them.