In Magical Girl Lyrical Nanoha A's, when the masked men take on Nanoha and Fate’s identities, parts of their barrier jackets are not the correct color. This is likely because cats are colorblind.
In Fullmetal Alchemist, Hughes is able to realize that Maria Ross is being impersonated by Envy because she doesn’t have her mole. This doesn’t save him from being killed, nor does it save Ross from being framed for his murder in the manga.
In Naruto, a Rain Ninja impersonating Naruto puts his shuriken holster on the wrong side (having a different dominant hand) and doesn’t include the cut Naruto got from Anko. Sasuke says this lack of attention to detail makes the Rain Ninja worse than Naruto.
In a filler arc, Naruto, Kiba and Hinata invoke this trope to distinguish themselves from their impostors. They have Hinata, who injured her leg, stay behind, and have the real Kiba be identified by having Akamaru with him, and the real Naruto identified by the chest. Amusingly enough, at the end of the arc, Naruto sets down the chest for a moment and Hinata, noticing he doesn't have it with him, sends him flying with a palm strike.
In another filler arc, the villains impersonate Hinata, but the fake Hinata doesn't blush when coming into contact with Naruto.
In an earlier arc, Orochimaru masquerades as Naruto in order to infiltrate Team 7. When Sasuke asks for the password, Orochimaru!Naruto says it without problems. Sasuke later states that Naruto would have never gotten the password correct on his first try.
In the Sasuke Retrieval arc, Shikamaru disguises himself as Jirobo and pretends to catch up with the rest of the Sound Ninja Four. When Tayuya scolds him for being late, he apologizes, but Kidomaru refuses to hand over the barrel containing Sasuke, saying that Jirobo would have scolded Tayuya for her bad language.
In Bleach, Szyael makes clones of Uryu, Renji, Dondochakka and Pesche. Uryu and Renji’s clones’ hair is slightly different and they have markings on their eyes. Dondochakka’s clones have no spots on their backs. Pesche’s clones wear trousers rather than a loincloth. Szyael claims that this is not carelessness as much as modifying them to fit his tastes, and while Renji claims he is stupid for doing this, Uryu notes that Renji didn’t notice until he pointed it out.
In Tsubasa Reservoir Chronicle, a minor antagonist tries to protect himself from Syaoran's attacks by claiming to have taken Sakura hostage. However Syaoran realises that the Sakura in front of him is just an illusion, since she calls him "Syaoran" instead of "Syaoran-kun".
In Detective Conan, there is a flashback episode where a grown up Shinichi is combating Kaito Kid (who can near perfectly disguise himself as anyone). Shinichi figures out which police officer Kaito was disguised as because he was the only one who remembered his own I.D. number. No one remembers their I.D. number.
In Kinnikuman, Terryman attempted to masquerade as Kinnikuman to fight the Seven Devil Choujin while Kinnikuman recovered from his first bout with SteCasse King. He's quickly busted by being left-handed when Kinnikuman is right handed.
Marine Boy: A false Marine Boy calls the real one's Dad by a variation of "father" the real Marine Boy doesn't use and this clues Marine Boy's Dad to the fact someone is impersonating him.
The Spider-Man mini-series Spider-Man: Hobgoblin Lives!, Peter and Mary Jane are conversing about Ned Leeds, the man they all thought was the original Hobgoblin. However, as they were doing so, Peter realizes something he should have had ages ago: if Ned Leeds was the real Hobgoblin, the assassins that came in and killed him would have been the ones dead because he would have had the Goblin Formula in him. That leads to everyone realizing they'd been had.
In The Smurfs comic book story "The Fake Smurf", Gargamel (and Hogatha in the Animated Adaptation) was capable of creating a formula that can change him into the form of a Smurf...but one detail that was missing in the transformation was that his impersonation doesn't have a tail. Gargamel tries to correct this by gluing a fake tail onto his behind, but later on the tail ended up falling off, and Gargamel was soon spotted and revealed to be the fake Smurf.
Early in The Superior Foes of Spider-Man, Boomerang figures out that the gangster Hammerhead menacing him is actually disguised Chameleon, because Chameleon!Hammerhead was talking in a stereotypical gangster accent, and while that is Hammerhead's signature trait, the real Hammerhead had actually dropped the quirk at that point.
In the Fresno, California-based Doctor Who Audio Dramas' continuity, the DavidSegalDoctor traveled with mute reporter Susie Jo Parker. Although mute, Susie Jo did speak. Usually when an impostor took her place and didn't research her very well.
In White Devil of the Moon, the impostor, posing as Reinforce, referred to Hayate and Signum as 'the two of them' when the real Reinforce would have noticed that Signum was in Unison form, indicating the presence of a third person.
In the Pony POV Series, Twilight attempts to repair Trixie's broken psyche, only to find herself with a psychiatrist who claims that she suffered a severe mental breakdown after accidentally killing Trixie completely and dragging the corpse around pretending it was still alive. Twilight starts to have suspicions when the doctor tells her that her "three friends" dumped her there, and moreso when the doctor can't understand why Twilight would think the Princess would care about this. Twilight is actually trapped by Trixie's subconscious, and Trixie never met Fluttershy or Pinkie, nor did she know about Twilight's official position.
In Waking Nightmares, Soldier, Derpy, Dinky, and Amethyst Star get replaced by changelings. The deception lasts less than a day before the protagonists wisen up to the inconsistencies (because the real Soldier never calls Spy a pervert [coward, sometimes maybe, but not pervert], Derpy hates English muffins, and Amethyst hates the Doctor due to some unspecified incident) and lead the imposters into an ambush.
In the original Shadowchasers, Jinx is looking for the shapeshifting boggart in a wax museum (an incredibly dangerous situation, like looking for a needle in a haystack and the needle is trying to kill you). However, when she passes by a display representing the duel between Obi-Wan and Darth Vader in A New Hope, she notices that the Darth Vader statue has a green lightsaber, and realizes that must be him.
In Megamind, Megamind blows his cover in one scene by pronouncing Metro City to rhyme with "atrocity." He just barely avoids doing the same earlier in the film.
In The Sorcerer's Apprentice, sorcerers powerful enough to do so have the ability to morph into whomever they wish. When Drake Stone, a less experienced Morganian, attempts to trick Balthazar into giving him the Grimhold while disguised as Dave, he accidentally lets his British accent slip, giving himself away.
He also forgot a second detail: the black nail-polish on the nails... which pretty much just confirmed the falseness of the imposter!
In the movie Dead Again, a man claims to be the fiance of an amnesiac woman and he presents a glove as proof. The glove seems to match the woman's except for one problem: it's for the wrong hand.
In Harry Potter and the Goblet of Fire, Barty Crouch realizes that Mad-Eye Moody is actually his son when Moody licks his lips nervously, which is an established Character Tic for Barty Crouch Jr. Dumbledore figures out that this Moody is fake because the real Moody wouldn't take away someone after they had just lost someone.
After killing the Big Bad in the Total Recall (2012) remake, Doug wakes up in an emergency vehicle with Melina holding his hand. He then notices that she's missing the bullet wound scar on her hand, forcing his "wife" Lori to reveal herself (using a holographic device he used earlier) and attack him.
In The Master of Disguise during the climax, Devlin caught Pistachio in disguise as a henchman because he was still wearing part of his previous "Cherry Pie Man" disguise. That and he knows the henchman is not married.
Though they're not impersonating one person in particular, the British spies in Inglourious Basterds are given away by the way they gesture to the bartender for three more drinks. A true German would have used his thumb, index and middle finger to denote three, while the Englishman uses his index, middle and ring finger.
In Wild Wild West, Artemis Gordon's has a nearly foolproof disguise as President Ulysses S. Grant (the fact that the same actor plays both helps) but West quickly knows it's not Grant because he was wearing the wrong school ring.
In Maximum Ride, the clone meant to replace Max has little knowledge of the flock, which means she doesn't know that Iggy is the Team Chef despite being blind, while she is supposed to be a terrible cook. Her cover is blown by the fact that six-year-old Angel is a mind reader, and was never fooled in the first place.
This is the Fatal Flaw of the main antagonist of Needful Things. He cooks up fake evidences of crimes or slights and then plants them on people to turn them against each other, but he keeps getting small details wrong, either out of arrogance and negligence, or maybe for sport. For instance, the town's sheriff is shown an illusion of the death of his wife and child being due to a local hoodlum (in order to make him try to kill the man). The sheriff realizes it's a fake when he notices that they're wearing their seatbelts in the illusion - when not wearing them was what killed them in the first place.
The Faceless Men, a guild of master assassins in A Song of Ice and Fire, disguise their identities by a combination of glamour magic and acting skills. Part of their training, therefore, involves completely rejecting all traces of one's real identity, including name, family, and personality. Arya Stark is told off every time she gives herself away with the Character Tic of chewing on her bottom lip.
In Allen Steele's The Tranquility Alternative, one of the first cracks in the imposter's assumed identity is when he doesn't respond appropriately to an elaborate online *cough* "roleplaying" scenario - such as not knowing that his virtual lover's fictional husband was killed off years ago. (An author's aside notes that the original would have told his captors anything they wanted to know... but they never thought to ask.)
The former Trope Namer (as Impostor Jamie Has No Accent) is in the Doctor Who episode "The Faceless Ones", when Jamie has been captured by the Chameleons—and Chameleon!Jamie does not have his Scottish burr.
In the episode "New Earth", Lady Cassandra (who speaks in a rather posh fashion) takes over Rose Tyler's body. Aware that she doesn't sound like Rose, she asks her assistant Chip how she should speak to the Doctor, to which he replies "Old Earth Cockney". Hilarity ensues.
In "Tooth and Claw", the Doctor accidentally ends up in the Scottish moors. The moment he realises they're in Scotland he switches to a Scottish accent (actor David Tennant's natural voice, in fact) and pretends to be a Scottish physician named Dr. James McCrimmon so that Queen Victoria doesn't shoot him. Later at dinner he slips back into an English accent and Victoria calls him out.
In the original series story "The Android Invasion," the Doctor can tell Sarah is an android because the android Sarah is wearing a scarf while the real one wasn't, and real Sarah hates ginger beer.
The "Datalore" episode of Star Trek: The Next Generation made clear that Data doesn't use contractions, while Lore can. In an inversion, even though Data is the person Lore is copying, Data was the younger of the two, and so technically "lost" the ability to use contractions. (In a now-infamous mistake, the "real" Data used a contraction in the episode's final scene, and depending on who you talk to it either wasn't noticed or they didn't have time to do another take. This also launched a thousand, or at least half a dozen, wild mass guesses when it was noticed.)
In Red Dwarf the impostor Lister can play the guitar really well. The real Lister only thinks he can.
In the same episode, Imposter!Kryten calls Lister 'Dave', while the Real Kryten always calls him 'Mr. Lister'.
Tested in MythBusters when Adam and Jamie tried to disguise themselves as each other - despite much effort, vocal cues and accents ended up being the weakest part of the ensembles.
Although Adam did better impersonating Jamie than Jamie did impersonating Adam, in part because Adam frequently mimics Jamie for amusement. That may have worked against Adam, however; while his impression of Jamie was better, it had also been used on-camera in the past, so the test subjects were more likely to recognize the "Jamie" voice as an impersonation by Adam.
In the "body swap" storyline of Buffy the Vampire Slayer, Buffy had Faith's Bostonian accent on certain words. (No one really noticed in-universe.)
Nicely done in Merlin when Morgana resurrects Lancelot from the spirit world, brainwashes him, and sends him to Camelot in order to stir up trouble. However, Merlin catches him out by phrasing a sentence that casually references the use of magic - Lancelot being one of only two people in Camelot who knows that Merlin is a warlock. But because Morgana doesn't know, then fake!Lancelot doesn't know either, and he responds in a manner that makes no sense from one who should be aware of Merlin's secret.
Smallville: Since Earth-2 Lionel is biologically the same person, he thinks that he can fully get away with seizing control of LuthorCorp from Earth-1 Tess. The only thing that's different between himself and his Earth-1 counterpart is that their fingerprints are opposites of each other. Earth-2 Lionel realizes this and reissues papers so that his fingerprints are on the new record, but he forgot one thing: since in his universe Tess was never given up for adoption, he overlooks the fact that all of the papers Earth-1 Lionel left at the orphanage with her have his fingerprints on them. This allows Tess to prove that he's a fraud and regain control of the company.
A season one episode of Sanctuary involved Magnus and Will being trapped in a crashed plane with a shapeshifting abnormal. Magnus discovers it when it's masquerading as Will when she asks for coffee instead of tea. The real Will would know that Magnus hates coffee.
In Lost Girl, Inari shape-shifts into Kenzi and impersonates her after kidnapping the original. Bo exposes her after she casually eats peanuts, which Kenzi is allergic to.
In the season two finale of Alias, Sydney offers Francie - or rather Allison, the spy who was turned into a double of Francie before killing her - coffee ice cream. "Francie" accepts, confirming Sydney's suspicion that she isn't Francie (who hated the stuff). All hell proceeds to break loose.
In the season two premiere of Sleepy Hollow, Ichabod battles an evil Ichabod while rescuing Abbie from purgatory. The winner is about to leave purgatory with Abbie when he calls her "Lieutenant," as he always does... but he uses the American pronounciation, something the real Ichabod would never do.
The Wild Wild West's "The Night of the Colonel's Ghost" has Artie impersonating Ulysses S. Grant in a bid to flush out a not-as-deceased-as-previously-thought colonel who's looking for hidden gold. Artie-as-Grant lists the battles the colonel and Grant fought in and all goes well - until he mentions a battle that took place after the colonel faked his demise.
The traditional Japanese greeting "moshi-moshi" is a version of this. Shapeshifting youkai wouldn't be able to pronounce it right, giving themselves away.
In Saints Row: The Third, The Boss attempts to impersonate Cyrus, the leader of STAG, but pretty much fails to act appropriately in any way. Given how poor the act is, it takes The Boss a surprisingly long time to get caught.
For a period of time in between major updates, Spies in Team Fortress 2 suffered from this when disguising. Unlockable weapons for some of the other classes had been introduced, but Spies taking the disguises of other players of that class would only show stock weapons. Unlocks such as the Ubersaw and Blutsauger for the Medic or Backburner and Flare Gun for the Pyro were astoundingly popular choices and anyone not using them was suspect. For instance, Medics who appeared carrying basic Syringe Guns were immediately suspected as Spies, making the disguise even more difficult to use convincingly. This disguising oversight has since been patched out.
A trait of a poor spy would also be always having their disguise carry the default weapon; its very odd indeed to see a Medic carrying a syringe gun over the medi-gun, or an enemy spy not to have a knife out. note This switch can be made by changing to your secondary and pressing "B".
Besides the above, there was one glaringly obvious tell once attached to the Medic disguise that made it completely unusable at the game's launch (which has long since been patched): When looking at a Spy-disguised-as-Medic, the "Ubercharge Built" meter didn't show up in their namebox like it would for a real Medic.
Spies disguised as Scouts are also prone to "forgetting" how the latter class moves; Scouts are noticeably faster than other classes and can double-jump, which disguised Spies cannot.
At one point in Bayonetta, the title character has to save Cereza from a giant Beloved angel. When the angel dies, Cereza falls into Bayonetta's arms... or, rather, the arms of a Joy angel mimicking her. The only giveaway? Angels can't hide their halos. A second giveaway is the fact that the Joy is fully clothed, despite Bayonetta being in the middle of a demon summoning at the time.
In order to get into the black market in Ueno in Shin Megami Tensei IV, you have to say "Erewan" (in fact, the player has to type it into the game). Presumably this is a shibboleth designed to root out demonic infiltrators.
Your clone in the Citadel DLC for Mass Effect 3 has more sort of forgotten every detail beyond "has the Normandy" and "wears an N7 badge"; doesn't have the charisma, doesn't have the flexibility, doesn't have the compassion, doesn't have the cybernetics, doesn't have a clue. You can actually point out, during the final confrontation, that there is no way the imposter is going to be able to fool, say, Admiral Hackett for even a minute.
The imposter did, to their credit, remember that despite being a clone they would need to replace the original's recorded fingerprints with their own, and arranged a complex scheme to do just that.
Star Trek Online: The side mission "Standoff" has the True Way try to frame a loyal Cardassian Self Defense Force gul as a traitor to Cardassia. The video that makes up part of the incriminating evidence turns out to actually be of a changeling in Gul Antos' shape, but the changeling got Antos' dominant hand wrong.
In Jackie Chan Adventures, Jade is able to spot Paco as a magical clone because he pronounces her name correctly, unlike the real Paco, who always pronounced it "Yade." Truth in Television. There is no equivalent to the English "J" sound in the Spanish alphabet (their "J" sounds like an English "H"), so many Spanish-speakers even those who are basically bilingual still talk about watching Yaws or Yudge Yudy.
In another example, in the episode P.O.O.L, Numbuh Four's friends are all replaced by evil twins from a negative universe originating from the pool. Hilariously, Numbuh Four fails entirely to spot that Numbuh One and Lizzie are fakes despite them acting very much like villains...but when he discovers that Numbuh Three has mean-looking eyes, that clues him in that his friends must be evil twins from another universe, because Numbuh Three would never look like that. Numbuh Four calls this deduction "being a friend!"
More specifically, Fake!Cadance doesn't recognize the rhyme that she and Twilight used to sing; this is the first clue Twilight gets that something is wrong.
In the IDW comics, a Changeling mimicking Derpy Hooves forgets to do the Fish Eyes. Another one has to smack it to make the eyes cock correctly.
In the episode "Too Many Pinkie Pies", in order to separate the real Pinkie Pie from her army of duplicates, Twilight Sparkle and friends round them up and make them watch paint dry. Any one of them that gets distracted is destroyed - the real Pinkie Pie is the only one willing to watch something so boring for so long, since she would rather be with her friends in the future than have fun now.
Episode "Spike at Your Service" features Spike doing favors for Applejack because she saved his life from Timberwolves. The girls decide to stage a fake Timberwolf attack so that Spike can "save" Applejack from doom. This didn't work because they forgot the bad breath that they have. Then the real Timberwolves show up and Spike end up having to save Applejack for real.
On Star Wars: Detours, the Decoys are supposed to look identical to Princess Leia in every way, but they're missing her most defining feature.
In Spider-Man: The Animated Series Spider-Man is able to figure out that Nick Fury is actually the Chameleon in disguise after realizing he has his eyepatch over the wrong eye. (The Chameleon had gotten the disguise by looking at Fury's picture in The Daily Bugle, where a negative had been flipped.)
Zigzagged in one episode of Ed, Edd n Eddy (it happens, but the imposter still fools the victim). At the end of an episode, Sarah and Jimmy use a two-person costume to dress up like Eddy's brother in order to scare him, and then tell him to get them ice cream. Eddy is at first a little suspicious because his brother is lactose intolerant, but Sarah's acting is so convincing that he's scared into complying. Eddy's reaction is, or course, a Foreshadowing to the movie, where it's discovered how much he's really terrified of his brother.
In the Adventures of Sonic the Hedgehog episode "Tails' New Home", Sonic and Tails believe that they found the latter's parents and the former returns him to them. However, Sonic starts reminiscing about their first meeting and realizes he's been had - they kept calling him "Tails", not "Miles", his real name. Tails' "parents" were robots and he was captured by Robotnik.
In Johnny Test Dukey has to figure out which of two Johnnys are a robot. When they answer the first two questions perfectly, he asks a math question. One Johnny answers immediately, and Dukey blasts him. The reason being Johnny has to use his fingers when he's asked such a question.
Pinky and the Brain: When Brain decides to take over the world by impersonating Abraham Lincoln, he's not worried that people will notice that he doesn't sound like the real Lincoln because Lincoln died before voice recording devices were invented and Brain doesn't believe there's any living person old enough to have heard Lincoln talk. The oldest living American proves him wrong about the last part.
For example: the words "brânză" and "cârpă" were often used to distinguish native Romanian speakers from native Hungarian speakers. In both words the "â" is pronounced as a close central unrounded vowel, while the "ă" is a schwa. Neither of these vowels exist in Hungarian, and many Hungarian speakers tend to confuse them.
During World War 1, a British officer tried to fool an Askari of the German Schutztruppe in East Africa at night by giving him an order in Swahili. He got shot dead immediately because he could not disguise his British accent, specifically by the way English turns many long vowels into diphthongs.
During World War 2, the forces of the Ustasha regime in Croatia were able to detect young Serbs (who belong to the Orthodox church) by telling them to make the sign of the cross.
During the Battle of the Bulge, Germans in U.S. uniforms infiltrated the allied lines. One of the quick ways to check out an unknown soldier was to ask him to say "squirrel".
A similar test to spot Japanese saboteurs without coming close enough for a gander of their face was ordering them to say "Lollapalooza".
Impostor Forgot One Detail, Impostor Jamie Has No Accent