In Batman/Huntress: Cry for Blood, Huntress gets an old photo of her deceased mother, Maria Bertinelli, in flagrante delicto with mob boss (and rival to mob boss Franco Bertinelli, Maria's husband) Santo Cassamento, and demands to know what they were doing in that hotel room together. His answer: "Isn't it obvious? We were conceiving you, Helena Rosa." Even worse: when he was instructed to kill the rival mobs, the Bertinelli, he saw a chance to Murder the Hypotenuse, and instructed his henchman to kill all the family "except the sister" (meaning, María Panessa, sister of the mob leader Tomasso Panessa). But the henchman, who was not used to the usual mob words, saw a regular family (a man, a woman, a male child and a female child) and spared the female child, the "sister" (yes, Helena). You can be sure that, after hearing this, no threats or manipulations could possibly keep Cassamento alive for very long. The blood cries for blood.
The plot of Batman R.I.P. essentially involves multiple Mind Rapes piled on top of each other to see how many it takes to make Batman crack. The biggie? Enigmatic villain Simon Hurt claims that he is actually Thomas Wayne, and that he faked his own death after hiring Joe Chill to kill his faithless whore of a wife. The truth of his story hasn't been determined, but Bruce decides to reject it at the end of the arc.
In Booster Gold One Million, it is revealed to the audience — but not to any of the other characters — that Booster's mentor Rip Hunter is actually Booster's son.
In nine issues, Brightest Day has already had three separate revelations about familial bonds between heroes and villains.
In Green Lantern Corps #35, Sinestro tells Soranik Natu that she is his daughter. He left her in the care of her foster family to keep her away from the dangers of being associated with him; making this one of the only semi-decent things he's ever done in his life. Since it's Sinestro, it's not clear if he's lying or not, but the last page of Green Lantern #36 makes it pretty obvious that he's telling the truth.
A Legion of Super-Heroes mini-series in the '80s started with the premise that R. J. Brande, the team's billionaire sponsor, was dying, and in order to save him, the Legionnaires had to figure out which one of them was secretly his child — an idea that had never been even hinted at before. It finally turned out to be Durlan shapeshifter Chameleon Boy; Brande was revealed to also be a Durlan who'd contracted a disease that froze him into human form.
Rose Walker's grandfather in The Sandman is Desire, who impregnated the comatose Unity Kinkaid during Dream's imprisonment.
Subverted with super-villain Lesla-Lar. After being disintegrated, her consciousness lived on. This messed with her mind to the point where she thought Supergirl was her sister. When she made this claim, Kara replied she had no siblings.
Buzz: Dominique... I am your father... Oh, I'm certain that didn't sound too "Darth Vader".
In Supergirl vol 6 #24, super-villain Cyborg Superman gets his memories back... and remembers what he is Supergirl's father. Several months later, in Supergirl vol 7 #2 he tells Kara he is her father (who she had previously thought dead).
Chris, Superman's adopted son, was found in a rocket similar to his. It was revealed during an invasion that the rocket was sent from the Phantom Zone, and he was the son of Zod and Ursa.
Lilith from the Teen Titans knew nothing about her past. Nothing at all. Until her mother kidnapped her, to rule Olympus together. Her identity? Oh, nothing important: it was Thia, a Titan of Myth and goddess of the sun. Which implies that Lilith is a demigoddess.
Wonder Woman Vol 2: Diana is quite distressed when Ares informs her that he's her grandfather, given their long history of fighting each other and Ares' very violent nature. Ares himself only recently learned of the fact, as his parentage of Hippolyta had been hidden from him. While Ares never becomes a "good" guy, he does occasionally go out of his way to prevent Diana's death due to this revelation.
Pre-Flashpoint, (Vol 3) Diana was told that one of the Hechatonkaires was her "father": the clay that was used to create her came from his imprisoned body. Whether or not this counts is up for debate, but the Hechatonkaires in question latched onto the idea as another reason to hate her.
In The New 52, she learns that the whole clay story is a lie, and she's the daughter of Hippolyta and Zeus.
Cassandra Cain was "raised" by the assassin David Cain, never knowing the identities of her true parents. In the final arc of the Kelley Puckett run of the book, she finally comes to the realization that Cain was her real father, but her mother's identity is left unknown until the final arc of the entire book, where she confirms, as she has suspected for some time, that Lady Shiva is her mother. Her Post-Flashpoint counterpart knew that David was her father the entire time, but it is only during the League of Shadows arc in Detective Comics (Rebirth) that Shiva reveals that she is Cassandra's mother, sending Cass into a Heroic BSoD.
Dark Nights: Metal: The demon Barbatos claims that he manipulated Bruce Wayne's entire life to get him on the path to become Batman. He claims this makes him Batman's true father.
Watchmen has a particularly twisted example of this. Laurie, the second Silk Spectre, knows from a tell-all book written years earlier that the recently-deceased Comedian tried to rape her mother, the first Silk Spectre. Then Laurie finds out that after the near-rape, her mother had consensual sex with him anyway, and that the Comedian is in fact her father.
Eighteen years after their creation, it was revealed that AvengersScarlet Witch and Quicksilver were actually the children of their former leader, long-time X-Men antagonist Magneto. Their feelings over this revelation were mixed, to put it mildly. Still, as Magneto is a Drama Queen, he did not told this directly. He told that Luna was his granddaughter, and let them react to that.
Polaris, of the X-Men, was revealed early on to be Magneto's daughter, too. But then that was revealed to actually be a plot by the villain Mesmero, who was using a Magneto robot to make that claim. Then later it was again revealed that she is his daughter... then once again not. Currently, she is once again revealed to be Magneto's daughter... ugh.
Hiro-Kala knew that he was the son of Incredible Hulk. He didn't know that Bruce Banner was the Hulk. So when they first met, the following exchange happened:
During John Ostrander's run of The Punisher, Jigsaw has kidnapped the member's of a mafia family that Frank has fallen in with. While talking with the oldest sister, he tells them why he's doing this; he and Frank are cousins. When they were kids, Frank was always showing him up, and everyone loved him. When she asks him if it's true, he says nah. He was just lying to pass the time.
A pivotal turning point in the original Spider-Man 2099 run featured Miguel O'Hara (Spider-Man) discovering that the main antagonist of the series and head of the megacorp Alchemax, Tyler Stone, was in fact his father. This derailed the current plotline for quite a few issues as Miguel wrestled with his hatred of the man. The trope was lampshaded near the end of the title's run, when Tyler attempted to shock Miguel into following his orders by dropping the bombshell of his parentage, only to have Miguel, who by this point had come to terms with the fact, casually sip his coffee and then smugly and calmly reveal that he knows.
Tyler Stone: And you will do it... because I'm your father. Miguel O'Hara:[sips coffee] Yeah, I know. Tyler Stone: You... you know? Miguel O'Hara: Yeah. Now get out of my office... dad.
Mr. Sinister pulls a particularly unpleasant Luke, I Am Your Father on Gambit in X-Men: The End when he reveals that Gambit is a clone created from Sinister's original DNA mixed with that of Scott Summers. Thankfully, this probably isn't canon — the Summers have a messed-up history as it is.
Speaking of the Summers family, several years after his introduction, Cable was retconned as being Nathan Summers, Cyclops' baby son who he'd previously been forced to send into the future to save his life. What happened was in the Bad Future Baby Nathan grew up oblivious to his parentage and eventually time-traveled back to the present, though by that point he was older than his father.
In their Star Comics' imprint Planet Terry series, the Big Bad Vermin the Vile gets Terry, who has been searching for his parents throughout the galaxy, to believe that he is actually Terry's father. However, it is later revealed that a villain known as The Hood, who is about the same age as Terry, is actually Vermin's real long-lost son.
The original Gen¹³ had more of a "Luke You Are His Father", with Burnout and team mentor Lynch simultaneously learning that the former is the latter's long lost son.
Gen-Active revealed A. Sublime of DV8 isn't adopted, and B. her biological father is Michael Cray, alias Deathblow, one of the most prominent figures in the Wildstorm universe. Notably, it's revealed to the reader, but not necessarily the characters — Sublime's mother doesn't seem to remember his name.
In the first G.I. Joe comic series, a boy named Billy living in the Cobra-controlled town of Springfield had joined the local anti-Cobra underground, believing that his father had been swayed into joining the organization by Cobra Commander. He'd failed to realize that his father was Cobra Commander all along. Destro knew the truth, however, and revealed it when he intervened to stop Billy from assassinating the Commander.
Knuckles' girlfriend Julie-Su discovers that she's the half-sister of Dark Legion leaders Kragok and Lien-Da, which also makes her a descendant of Knuckles' ancestor Dimitri (making her and Knuckles very distant cousins). And sometime later, it's revealed that Remington is Kragok's son.
In a dark alternate universe, 30 years in the future, Lara-Su was raised to believe her father Knuckles had been killed by Constable Remington. However, after she goes back in time (and travels to the wrong dimension too), her mother Julie-Su finally tells her that, in fact, Knuckles wasn't killed by Remington, but went insane with Chaos Powers and became the dreaded leader of the Dark Legion and by extension tyrant of the entire planet, Enerjak.
Star Wars: Darth Vader shows the inverse in issue #6: Boba Fett reveals to Vader that the boy who destroyed the first Death Star was named Skywalker. After Fett leaves, there's an Internal Reveal flashing back from the end of Revenge of the Sith to a little after A New Hope (to Luke and Vader's first confrontation in a previous comic). Vader is mostly silent, but his hand is shaking with enough Force energy to shatter the glass window in front of him.
Vader: Skywalker. [Later — Vader folds his arms. Scenes flash in his memory. Padmé crying. Padmé's funeral. The destruction of the first Death Star. His first confrontation with Luke.] Vader:I have a son. The glass before Vader is almost completely gone now. Vader: He will be mine. It will all be mine.
At the end of the canon comic series Darth Vader: Dark Lord of the Sith, Vader undergoes a series of dark side visions relating to his past, present and future. One of these visions heavily implies that Palpatine himself was responsible for Anakin's conception through the Force (and thus tying back to the revelation that was planned to originally occur in Revenge of the Sith).
In Issue 6 of the Dallas arc of The Umbrella Academy, it's revealed that Number 1 and Number 5 are biological siblings (they were adopted siblings before this point).
Tossed in a blender with Luke, You Are My Father (but not really) in Usagi Yojimbo: Usagi discovered that his old flame Mariko's son Jotaro is actually his son, and Mariko's husband (Usagi's very unfriendly rival) also knows but loves Jotaro anyway. Usagi and Jotaro's sword master guessed almost immediately (they're very much alike), and gives them the opportunity to travel together for several months; after talking with Tomoe about responsibilities and relationships, Usagi decides not to break the bond between Jotaro and his family, while Jotaro decides not to force Usagi to give up his wanderer lifestyle to take care of him. It turns out Mariko told Jotaro the truth, but left out the part about Usagi knowing too. Upset at his "weakness" and the fact that he doesn't know when or if he'll ever see his "uncle" again, Jotaro calls out "Miyamoto Usagi! You are my father!!" but Usagi is already too far away to hear ("Just a trick of the wind, I must be tying my ears too tight"). TL;DR: Father A and Son B know they're related, but they don't know the other knows the truth (C,D,E, and F know the truth too but they aren't talking, mainly out of respect).