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- Nenene and Toki from Code:Breaker both have one blue and one brown eye. This almost gets Nenene killed when fire controller Oogami mistakes her for his despised teammate Toki who is Nenene's long-lost brother. Nenene does not seem to notice the resemblance, even when Toki is standing in front of her while Sakura says "This is your brother!"
- In Fullmetal Alchemist, the fact that Ed, his brother Al, and their father Hohenheim all have gold eyes and gold hair (not blond, gold) is an important plot point, as it marks them as ethnically members of the lost Xerxes civilization. Hohenheim is an immortal survivor of the apocalypse. The first anime missed the memo and gave Al blue eyes, but it still mostly works.
- The Count D from Pet Shop of Horrors has heterochromia, one eye being purple and the other, whom he hides under his hair, is golden. In the manga, it's explained that this is meant to be representative not only for his non-human nature, but also for his inheritance: his father and grandfather have golden and purple eyes, respectively. Since each one has a very different personality, and D has personality traits from both, his heterochromia symbolizes this dual nature.
- Rozen Maiden: Suiseiseki has red and green eyes, and was originally nicknamed "Boatlights-chan" by fans before she became known as simply "Desu". Her twin sister Souseiseki, introduced later, has the same eye colors but reversed. Appropriately, they are foils for each other.
- Luki and Noki from Dogs: Bullets & Carnage have mismatched eyes (gold with a black iris, black with a gold iris, with the actual which-eye-is-which swapped between twins). They're also both powerful cyborg Child Soldiers.
- Big Bad (of at least one arc) Rido Kuran of Vampire Knight has one blue eye and one crimson eye, a trait that shows up in the people he possesses. This is the first clue to Rima Touya that something's not quite right about Shiki...
- In Claymore, every claymore warrior for the most part looks human, however their one distinct feature, least before they start tapping in their Yoma powers, is their silver eyes. In one episode the main character, Clare, who's a claymore is task with hunting down a Yoma in a city that doesn't allow claymores. To get into the city she takes a pill that limits her powers but make her appear human just by turning her eyes brown.
- Lyrical Nanoha:
- Vivio from Magical Girl Lyrical Nanoha StrikerS has red and green eyes because Sankt Kaiser Olivie Sägebrecht from whom she was cloned also possesses those features.
- Magical Girl Lyrical Nanoha ViVid introduces Einhart Stratos, a descendant of another emperor from the Belkan Age, the Supreme King Claus G.S. Ingvalt. She also has heterochromia, albeit blue and violet eyes.
- In Samurai Champloo, the villainous brothers Denkibou, Umanosuke and Toube all share pink sclera (their eye whites) on their left eyes. It's arguably the only physical trait they have in common.
- In Tengen Toppa Gurren Lagann, some character's pupils (specifically: Lord Genome, Kittan, and Simon) take on spiral form to indicate their mastery of the Spiral Energy.
- Misaki Shokuhou from A Certain Magical Index has stars in her eyes. The same stars appear in the eyes of those who she's using her mind control powers on.
- Certain ninja clans in Naruto will have this. Most consistent are the Hyuga, all of whom possess white eyes called the Byakugan. Other examples include the Uchiha and their Sharingan, and the overweight Akimichi clan.
- Kakashi and the Big Bad Tobi both have mismatched eyes, with one of them being a Sharingan - turns out the Big Bad is Obito Uchiha, whose left eye was transplanted to Kakashi when they were children! Side-effects include shared vision when they're near each other and having the same Mangekyou Sharingan, Kamui. Due to Mangekyou's power it is safe to say Obito would not have been beaten without Kakashi's presence. Good thing Obito gave him that eye all those years ago!
- In Dragon Ball, all Saiyans are born with black hair and eyes. This was one of the reasons why Vegeta didn't believe Trunks was of Saiyan blood since he had blue eyes and purple hair.
- Two of the characters, a Granddaughter and Great-granddaughter of two of the protagonists - Lucy and Sarah Edmontons, in Bringing Me To Life share heterochromia. One eye is dark-gray, while the other is dark-brown.
- In one film version of The Hound of the Baskervilles, Sherlock Holmes realizes that Stapleton is a member of the Baskerville family because both he and a portrait of patriarch Hugo Baskerville have webbed hands. (In the original novel, Holmes points out that Stapleton is facially a dead ringer for the portrait of Hugo when you block out of view the antiquated clothing Hugo is wearing.)
- X2: X-Men United: Jason Stryker has differently-colored eyes, as does the illusory little girl he creates.
- The Pact: Annie Barlow has one green eye and one blue eye. So does Charles Barlow, implying Annie's basement-dwelling serial-killer uncle is also her creepy incest-dad.
- In A Brother's Price, all the princesses can be easily identified by the "royal red hair". There is also the Porter family, some of whom have eleven toes. The fact that Kij (who doesn't have the trait) has a daughter who has eleven toes is proof that the daughter was fathered by Kij's brother, Keifer Porter, who has the trait, and not, as Kij claims, by some anonymous man she had a one-time sexual encounter with.
- In Dragon Bones, there is a certain shade of blue eyes that is exclusive to members of the Hurog family. Ward, who doesn't have them, nevertheless immediately concludes that Oreg, the local Friendly Ghost and the Genius Loci of castle Hurog must be somehow related to the family, as Oreg has the Hurog-blue eyes.
- The horribly inbred Martense clan from The Lurking Fear by H.P. Lovecraft all have this as a distinguishing mark. They have brown-and-blue eyes apparently. This is the tip-off that the swarm of Humanoid Abominations haunting the area actually consists of their offspring.
- All the clone "shatterlings" of Gentian Line in "House of Suns" by Alastair Reynolds have one green eye and the other a "wintery jackdaw blue", since they're all male and female "facets" of Abigail Gentian. it is noted that since they have a bit of leeway from Abigail's exact appearance, in some shatterlings the mismatched eyes are toned down so much as to be unnoticeable.
- In A Song of Ice and Fire, certain members of House Targaryen have a physical tolerance to high temperatures and fire; also, they all share a characteristic light-silver hair and violet eyes, a trait of the peoples of Old Valyria.
- One Goosebumps book has the main character's best friend be a girl with heterochromia. This becomes a plot point when she mysteriously disappears and he starts seeing a dog with heterochromia around the neighborhood.
- The generational sagas by author Edward Rutherfurd often use physical genetic quirks, such as webbed digits or Skunk Stripe hair, to illustrate a bloodline's continuity down through the centuries.
- In Warrior Cats, at one point Hollyleaf notes that Cinderheart always flicks her paw a certain way when grooming herself. She notices Leafpool staring at Cinderheart, and Leafpool comments that Cinderpelt used to flick her paw like that. Cinderheart is a reincarnation of Cinderpelt, and while the reader already knows this, it's one of the first times the characters see the resemblance between the two.
- In The Godfather, Sonny Corleone has a very large penis and can only have normal sex with Lucy Mancini, who has an abnormally large vagina.
- On Kyle XY, one of the first things that tips Kyle off to Jesse's connection to him is the fact that neither of them has a belly button.
- In the Murdoch Mysteries episode "Belly Speaker", the victim and his son (who's a ventriloquist) both have mismatched eyes, one blue and one brown. The son's Demonic Dummy, modelled after him, has mismatched eyes as well. It's later revealed that he had a twin brother who also had such eyes.
- One episode of Forever used this as a major plot point: a diver that had died in the expedition to find a sunken ship had heterochromia - one green eye, one blue. This tipped off Henry to the fact that the bartender who served drinks to the victim the night of his death was the sister of the dead diver, as she had the same eyes, revealing that she had killed the man she felt was responsible for her brother's death.
- The play Redwood Curtain is centered on a young Vietnamese girl who was adopted by an American family. She has "eyes like a husky dog" (one blue, one dark). When she meets a homeless Vietnam vet who has similarly mis-matched eyes, she starts wondering if he could be her biological father. He's not, but he knows who is.
- Mortal Kombat: Armageddon: The protagonist of Konquest Mode, Taven, is shown to be related to his brother and antagonist Daegon by a dragon tattoo on their faces, which are inverted mirror images of each other.
- In Tales of Graces, this is a sign that Lambda is inside somebody's body. It happens to Richard and later Asbel, and this carries into Tales of Graces F. When Lambda is inside Richard, his other eye is red, but when it happens to Asbel, it's actually purple, signaling that Lambda has a different relationship with Asbel than Richard.
- In Devil May Cry 3, Lady and Arkham and Jester both have one blue and one red eye. This serves as a visual clue that they're father and daughter.
- Iris and her twin sister, also named Iris, from Rune Factory both have green and red eyes. They used to be one woman but were magically split into two entities.
- In The Witcher 3, the Voluntary Shapeshifer Dudu is missing an eye, and as a result every form he takes is also has a closed eye with a scar over it.
- In Fate/stay night, homunculi made by the Einzbern family have distinctly silver hair and red eyes. This applies to Ilyasviel; her mother, Irisviel, and the homunculus on which the two former are based, Justeaze.
- Christian Weston Chandler in Sonichu has heterochromatic eyes, though it's not at all striking. It's a feature of his Evil Twin (with inverted colors) and his Chris-Chan Sonichu form, too.
- When Abel of Dan and Mab's Furry Adventures first enters the Succubus and Incubus Academy, he attracts interest because of his heterochromia and the dual batlike/birdlike nature of his wings. Both of these are traits of Quoar and his elusive clan members.