This is based on opinion. Please don't list it on a work's trope example list.
Tear Jerker / Wander over Yonder
I'm just a little black cube of darkness...
In "The Fugitives", the heartbreak on Wander's face when he thinks Sylvia's abandoned him.
In "The Good Deed", Wander is so disheartened by his failed attempts at helping others that he lets himself sink into the mud as a missile heads for the sun.
In "The Pet", Sylvia thinking that Wander's been killed by the alien, when what she found was really the stuffing to a teddy bear given to the alien by Wander
And when she mourns him, she recites what he would have to say to her: "It's always sunny somewhere, Sylvia old girl. So keep your eye on the horizon and don't look back. Never look back!"
In "The Box", Wander thinking he has let Sylvia down for not believing he can overcome his temptation of opening the box.
In "The Hat", Sylvia spends most of the episode being so sure she knows where Wander is, and when she finally gets there, after receiving a call from a drowning Wander, it turns out she's wrong. And she's just devastated, falling back in despair, feeling she's too late to save her friend. To make matters worse, the phone call was just Wander telling her he'd always love her, and then a gurgling, barely-heard "goodbye" as water covers his head.
Huckleberry Knucklehead running off crying after hearing Wander and Sylvia arguing in "The Toddler" can be a little gut-wrenching... especially when you realize that he was voiced by an actual toddler (Lauren Faust's nephew, to be exact).
In "The Rider," when Wander thinks that Sylvia would rather be partners with Ryder than him.
Wander in 'The Helper', trying SO hard to make people happy, and being the biggest example of 'Don't set yourself on fire to keep others warm'
Peeper's heartbreak when he learns that Lord Hater prioritized destroying Wander over the "No Planet Left Behind Bombapalooza" after promising to kick the habit. He interrupts the "wedding," and breaks down into Inelegant Blubbering saying, "I thought we had something special!" It's not helped by Lord Hater saying that while Peepers is right, he doesn't care about Peepers's feelings.
When Wander has to drive Lord Hater away from him and Sylvia in "The Fremergency Fronfact", the entire sequence is heartbreakingly gut-wrenching... especially if you had to deal with this exact scenario before.
Keep in mind that Hater basically has the mind of a small child here.
A minor one but in "The Boy Wander" we have Wander getting beaten up by Screwball during the latter Villain Song
The ending of "The Wanders" teaches us something about Wander. "The helper seeks to help, because he knows what it is to be helpless." And Incomplete!Wander visibly recoils when Sylvia tries to re-merge him with Helpless!Wander. He also seems to be VERY insistent on leaving without the last Wander and claims that he's "better off without him". Just what exactly happened to him that made him fear feeling helpless so much?
Another, more subtle moment in "The Wanders". When Sylvia refuses to merge Contrary!Wander, all the Wanders in the cave are visibly distraught. You can tell that they're upset that their best friend doesn't accept them for who they are.
In "The Axe", after Peepers is fired, there's a surprisingly heartbreaking montage set to a sad song where Peepers is looking around the ship and remembering all the fun times he had with Lord Hater.
Hater takes Comedic Sociopathy to the next level when he forces Peepers to delete all the photos of them together from the ship's computer system. Funny in kind of a tragic way.
Hater: Delete that. Delete that. Delete that. Delete that. Delete that. Delete that. Delete—Actually, no... These memories. We've been through a lot together, haven't we?
Peepers:(hesitating hopefully) Yes, sir. We sure have.
"The Black Cube" is one of the saddest, if not the saddest, episodes yet. After the humiliating incident in "The Battle Royale", the BLACK CUBE OF DARKNESS has hit rock bottom: he's stuck living in a seedy apartment, working a dead end job, his girlfriend left him, and everyone is either terrified of him or sees him as an object of ridicule. You will feel sorry for an eldritch, soul-sucking piece of Sinister Geometry after watching this episode.
Also, Wander's Heroic Sacrifice in the hands of the cube, who has now completely lost it and is lashing out at the crowd. Even though he survives, it's a pretty heartwrenching scene.
The way Hater keeps trying to Kick the Dog in "The Hole... Lotta Nothin'", It starts off as funny with all of the Call Backs and trolling, but then his methods suddenly start getting deep and off-putting...Luckily, karma gives the skeleton a huge shot to the chops at the end.
More specifically, he fakes a phone call to Sylvia and tells Wander she told him she never liked him and wasn't coming back for him (while it's doubtful that Wander actually believes this, he's deeply affected by the mere idea). He swiftly follows this up by pretending to be sorry for being an "insensitive jerk" and telling Wander he'll give up his evil ways… if Wander comes over and gives him a hug (which he can't, because he's stopping the hole with one arm). Finally he makes Wander question who he is if he can't live up to his name anymore: "You're just a whole lot of nothing." It's startling because it's so far removed from Hater's usual cartoony mission of "destroying" Wander—it's much more cold-blooded and personal. He doesn't want to kill Wander, he wants to break him.
"The Show Stopper", the chained-up bunny people are the first species that Wander failed to save from Dominator's rampage of destruction, due to him being distracted by Hater's rock show throughout the while episode.
Though Word of God claims that they managed to survive due to Dominator chasing after the main four instead of focusing on them.
Beep-Boop's Heroic Sacrifice in "The Bot". After betraying Dominator, he is ejected into space where he burns up in the atmosphere. As he burns, he takes one last look at the picture of him and Wander from the photo booth.
Beep-Boop: I... am... Beep... Boop.
After this, Wander and Sylvia sit on the dock at night and Sylvia mistakes Beep-Boop for a shooting star. She asks Wander to make a wish. Wander smiles and says "Good luck Beep-Boop." The next scene shows the photo of Beep-Boop and Wander silently drifting through space, with Beep-Boop nowhere to be seen.
The Stinger just piles it on, showing Beep-Boop's sand art of him and Wander being slowly washed away by the tides.
It becomes Harsher in Hindsight (as if it needed to be) later on because by the time of the finale, Dominator has destroyed every planet except the Secret Planet, which means his sacrifice was all in vain.
"My Fair Hatey" wherein Dominator basically crushes both Hater and Wander's spirit. First via by laughing at Hater's misunderstanding right in his face, then via her Villain Song wherein she explains that she's "the bad guy".
What makes this even more heartbreaking is the fact that this whole episode started with Hater actually asking Wander for help in his attempt to woo her, something that they both genuinely bonded over.
Even worse? The song right before this was "You're the Greatest", a genuinely heartwarming ballad Hater sings for Dominator. In it, he even states that he wants to rule the galaxy with her and that SHE was The Greatest in the Galaxy, something he's never called anyone besides himself.
This stanza from the titular song becomes Harsher in Hindsight in a meta way when you know that the show isn't being renewed for a third season. Almost sounds like Wander's singing it for us as much as he is for Hater.
"My Fair Hatey, don't you give up
It's not a total disaster
Hey, we gave it a go
Put on a heck of a show
And did a lot of fun stuff we'll get to laugh about after."
In "The Waste of Time", Old!Wander and Old!Sylvia spending 50 years on the same planet, and eventually disappearing after they prevent Wander from getting the Time Orbbles which led to the events of the episode happening in the first place.
Fridge Horror from the same episode: Wander has been confirmed to be much MUCH older than he seems-possibly even thousands of years old. And Word of God has confirmed that he never really aged much during the 50 years he spent with Sylvia-he was only wearing a fake beard and acting old to make Sylvia happy. In other words, Wander is most likely going to OUTLIVE Sylvia. This may well be one of the biggest tear jerkers in the entire series.
The ending to "The Night Out": Sylvia belatedly realizes that she spent the night with LORD DOMINATOR, an Omnicidal Maniac who's been destroying the galaxy and actually had fun with her. On the other side, Dominator has dismissed the idea of friendship and decides to continue her evil ways. The Power of Friendship is a definite No-Sell.
While it's portrayed in a comedic fashion, it's easy to feel bad for Lord Hater in the first part of "The Search for Captain Tim", especially for viewers who have also experienced having a missing pet. He desperately searches all around his ship and is rendered a heartbroken curled-up ball when he can't find Tim. Keep in mind, this is the same guy who hardly ever shows any sort of compassion for anyone else, which makes it even more upsetting. To rub salt in the wounds, the Watchdogs are happy that Tim is gone (though considering that Captain Tim regularly attacks them, it's not like their celebration is completely unjustified), so Hater has nobody to comfort him.
"The Flower" in its entirety. It opens with Wander and Sylvia viewing the devastated ruins of the galaxy they have thus far utterly failed to save from Dominator. The only sign of any hope is a single flower struggling to survive. The duo try desperately to nurture it back to health. For Sylvia, it's about keeping Wander from crying, because she is afraid that if Wander reaches that stage then that signifies that all hope of saving the galaxy is truly lost. The flower ultimately dies but, as Wander and Sylvia leave with their resolve still intact, the flower's spores spread over the dead planets and begin to grow..
It's very subtle, but when Dominator corners the duo, she refers to Wander by name, but can't remember Sylvia's. It's peculiar that she would remember the name of someone she has had little interaction with, but not the name of someone she had a night out with, right? Well, it's entirely possible that she is just pretending to not know Sylvia's name, as if to say that their night out meant nothing to her and that their "friendship" might as well have never even happened. Although considering this is Dominator we're talking about, it's also possible she genuinely didn't care enough to remember. Either way, it's heartbreaking.
Several moments in "The End Of The Galaxy", courtesy of Lord Dominator.
Wander's heartbroken cry when he thinks that Sylvia has been killed. Turns out it was a cruel trick by Dominator.
Wander's terrified, tearful expression when Dominator tries to impale Sylvia on her drill.
Remember, she considered herself friends with Sylvia just a couple of episodes ago, and now she's more than happy to kill her in the most brutal way possible. All so she can break Wander's spirit. It really hammers home how much of a complete sociopath she is.
Dominator mercilessly insulting Hater to throw him off his game. You can tell that she knows all his insecurities, and that she knows exactly how to get to him.
Dominator:"Please, even if you DO stop me, you'll still be a JOKE! The lamest in the galaxy! Just a dopey, self-centered jerk that nobody HAS EVER, or WILL EVER respect! Face it! You are literally the WORST. VILLAIN. EVER."
In this episode, we see Dominator at her most vulnerable. She's torn between her desire for companionship and her love for destruction. However, she is in complete denial about her loneliness, so much so that she covers her ears and threatens to destroy Wander when he simply brings it up, and would rather run off alone, with absolutely nowhere to go, rather than admit she is lonely. This episode makes it clear that as evil as she may be, Dominator still suffers from a great deal of emotional turmoil.
Just watch the scene where Wander talks to her as she's trapped by the drill. She's defensive, panicky and practically clawing for a way to escape. Wander has forced her into a corner, emotionally speaking, and to see her in such denial over her loneliness is honestly heartbreaking, especially to those who have been in a similar situation.
When Wander offers a second chance even after everything, the look in Dominator's face shows that for a brief moment, she legitimately considered it.
In The Stinger, even as she's cursing everyone else and calling them dorks, Dominator is sniffling as if she's about to cry.
There's also Slyvia silent reaction after Dominator ships explodes with Wander still inside, thinking that Wander died.