From the original Guardians, Stakar and Aleta's children being killed right in front of them. Adult Fear at its finest.
Later on, during an argument between them, Stakar breaks his usual stoic façade and starts breaking down when Aleta accuses him of not caring about their kids.
The flashback to Phyla's childhood showing that her mother wasn't exactly supportive or encouraging, and the implication that everything Phyla's done in life is an attempt to live up to her father's legacy, and that she feels inadequate compared to her brother Genis, i.e. the guy who went nuts and tried to destroy the universe. That's harsh.
The situation with Adam. After spending all that time trying to get back and save him, it turns out there's nothing left to save. He's been the Magus for months. And then it turns out his moment of Dying as Yourself was just the Magus messing with everyone's head to be a dick.
Phyla's for-real death. Just when Moondragon thought she'd gotten her back, she loses her again.
The team's situation after The Thanos Imperative. Phyla's gone, Adam's gone, Drax is gone, and Peter's stuck in a collapsing dead universe, meaning for all intents and purposes he's dead, and the rest of the team just split up and go their separate ways. Bittersweet Ending at its worst.
"Take my hand, Peter..."
Peter Quill's mother's death. Congratulations, Marvel. This is your first movie to rip everyone's hearts out in the first five minutes.
Young Peter is waiting outside of her hospital room. When given the news, he runs away because he couldn't bear to see her death. This hits home hard to those who have went through an experience like this. He is also listening to a mixtape she made for him, so his anger in the trailer when it's taken away from him is entirely justifiable.
Quill:[screaming at a guard while being tasered] That song belongs to me!
Her asking him to take her hand, complete with slow and obviously strenuous movement. He can't bring himself to, so he turns his head away from her... only for her to flatline while he's looking away. The way he says "No!" when he realises she's gone, and then starts SCREAMING and SOBBING for her to come back is just horrible. Downer Beginning, indeed. It's made worse when he's being abducted by Yondu. All he can do is scream for his mother.
Amplified even further in the Meaningful Echo at the end of the film; after grabbing hold of the Infinity Stone to keep it from Ronan, it begins tearing him apart from the inside out; Gamora then reaches out to him begging for him to take her hand. Peter momentarily finds himself standing at his mother's bedside again with her repeating her request from the beginning of the film.
The song young Peter's listening to outside the hospital room? 10cc's "I'm Not In Love." Which has an instrumental break (we don't hear) that features the repeated spoken phrase "big boys don't cry..." Ouch. Another example of how the soundtrack is just so damn awesome.
Hell, even the soundtrack can cause a few tears if you think about it. A few of the songs contain lyrics that can be interpreted to be Peter's mom comforting him over her imminent death ('Spirit In the Sky' with 'When I die and they lay me to rest/I'm gonna go to the place that's the best' and 'O-Oh Child' with '...Things are going to be easier/Someday we'll walk in the rays of the beautiful sun...')
You want an off-screen Tear Jerker? The reaction of Peter's grandfather when he discovers that his grandson is gone...
To make matters more depressing, apparently James Gunn initially planned for the film to end with the now-aged grandfather waiting for Peter to return, but was cut because viewers might not recognize the old man and that it was too sad.
He has this photograph of Meredith (Peter Quills mother) and Peter as a little boy and he looks up at the stars and we go up to the stars and it was really sweet. It means that he must have seen Quill getting abducted at the end of that day and is still waiting for him to return, but it was freaking sad, so we took it out.
Gamora's reaction to Quill's advances, while funny, is also very sad. The poor girl's been without "normal interaction with people" like flirting that her first response is to lash out and threaten someone with violence.
An intoxicated Rocket rants about how he's sure that everyone considers him to be a monstrous, freakish joke and implies that he was all too lucid when the scientists were experimenting on him.
Rocket: I DIDN'T ASK TO BE MADE! I DIDN'T ASK TO BE TORN APART AND PUT BACK TOGETHER OVER AND OVER! Turned into some... Some little monster.
Then we have the following:
Quill: Rocket, no one's calling you a monster. Rocket:[points at Drax] He called me vermin! [points at Gamora] She called me rodent! Let's see if you can laugh after five or six good shots in your freakin' faces!
Also notice how the camera briefly hovers on Gamora for a second during Rocket's rant. At that point in the movie, we had just learned Gamora's own origins; how her family was killed by Thanos, who then trained her (through torture) to be a living weapon. You can tell that Gamora instantly knows what Rocket is talking about.
Groot's delivery of "We are Groot..." followed by his sacrifice to protect the other Guardians as Ronan's ship crashes. While he may have begun to regenerate by the end of the film (or rather, his son), seeing him give his life for his friends truly is a Tear Jerker.
To add to the impact of that scene, right before he speaks, Groot uses a vine to wipe away one of the tears from a crying Rocket.
Rocket's reaction to Groot's sacrifice, as he, who previously treated Groot like a servant and an idiot, is devastated by Groot's death.
Rocket sitting by himself sobbing pitifully while clutching a handful of Groot's remains, looking smaller than he has for the entire film. It doubles as a crowning moment of heartwarming when Drax comes to sit beside Rocket and gently pet him on the head, earning a genuinely surprised and touched look from Rocket.
This is also a meta-example. This was the first role Vin Diesel took since friend Paul Walker's death.
Just, really, if you went to a movie theater and didn't see someone cry at Groot's sacrifice, it was because YOU were the one doing so.
In a exchange on Twitter on February 27th, 2018, James Gunn revealed that Groot did die and the sapling seen at the end of the movie and who appears in Guardians of the Galaxy Vol. 2, is actually his son.
The destruction of hundreds of Nova Corps ships is enough to even give Rocket pause.
Worse? As Saal is about to get crushed into paste, he calls Rocket "Rocket" and not by his experiment number.
Worse still? He's asking for help. Rocket, unable to leave the aerial defense of the capital, gets to helplessly listen to him die.
Kudos for Cooper too since Rocket actually replies to Saal's plead for help. He says "Saal, just hold on!" but not as a call for heroic willpower. Rather, the way Rocket says it is more like "Hold on, I'm coming for you as soon as I can." only for Saal to die shortly thereafter.
Quill finally opening his mother's last birthday present to him.
The last words of the letter contained within: "My little Star-Lord". Even more of a tearjerker is when you realize he hasn't been using the name due to an outsize ego, but in memory of his mother.
The first song on Awesome Mix Vol. 2 is Marvin Gaye's "Ain't No Mountain High Enough." Just think about some of the lyrics: My love is alive/Way down in my heart/Although we are miles apart.
Rocket: Stopping Ronan? It's impossible. You're asking us to die.
Blue Swede's "Hooked on a Feeling", Played for Laughs in the trailer, is used for some depressing Soundtrack Dissonance when Quill sees the prison he has just been put in. One of the prisoners can be seen sitting at a table and crying as she looks at a photo of her family. She's seen again just before Ronan orders everyone in the prison cleansed.
As if things couldn't get worse, Ronan arrives there and orders the entire prison cleansed. Sure the prisoners and guards were as far from good people as you get but there was the old man from whom Peter got the leg. The guy from whom Drax took his favorite knife. Rewatching those scenes is heart-wrenching because every single person in that prison is a dead man walking.
Gamora's words "Whatever nightmares lay ahead are dreams compared to what's behind me." And when she's in her cell, she's smiling. In other words, she considers being locked in a prison with hundreds of people who hate her the better option opposed to being with Thanos and Ronan. Think about that.
A minor one, but pay attention in the Collector's scenes and you'll notice a dark elf in one of the exhibits. The poor thing is huddled in on himself, trying to sleep despite the bright light. Horrible conditions for anyone to be kept in, but particularly cruel for a species that essentially lives in darkness. He's probably also one of the last of his kind.
In fact, since the Collector usually only collects rarities, he's probably in that cell because of his race's recent losses. Not many dark elves left, so now they're a... collector's item.
Notice also the rather apathetic looking Chitauri? Never thought you'd feel sorry for one of them? How about now...
Every intelligent creature trapped in the Collector's museum gets a raw deal.
The fate of Carina's predecessor is to be locked up in a cage, presumably indefinitely.
The fact that Carina was so desperate to get out of service to the Collector that she outright committed suicide by grabbing the Infinity Stone.
Just before Carina grabs the Stone and the whole place explodes, the captive in the cage behind her starts slamming her palms against the glass. Anyone's guess whether she's scared of becoming collateral damage, hoping to become collateral damage rather than remain a prisoner, or simply doesn't want Carina to die.
One of the less obvious emotional moments is watching Drax the Destroyer charge in screaming to save protagonist Star-Lord from Korath, the right-hand muscle of antagonist Ronan the Accuser.
Drax is a berserker on a sad quest to avenge his family's death at the hands of Ronan the Accuser. His quest is sad not only because Ronan far exceeds Drax in power, but also because killing Ronan will not bring Drax back his family.
However, Star-Lord enabled Drax to be part of a team, and in doing so gave Drax a kind of new family and thus a reason to fight and live, not simply fight and die. Thus, watching Drax charge in to save Star-Lord magnifies his character, making him more meaningful because he has grown to care for his allies and will bring his full strength and fury to bear to keep those allies from any harm.
Peter's insistence that he is 'Star-Lord'. Through much of the movie it comes off as him trying to create a nickname/code name for himself, something that he thinks is badass but to most comes off as weird and clunky. The ending reveals the origin of the name: it was the nickname his mother had for him. He wants to be called it because his mother is likely the only person who called him Star-Lord and he wants to hear it again.
And then he does hear it again, at the end... from Gamora, whom he'd previously envisioned as his mother while grasping the Power Stone.
A subtle Fridge Tearjerker hidden within the otherwise awww-cute dancing Baby Groot scene: Pay attention to what words from I Want You Back are sung over the scene:
Oh baby give me one more chance / (show you that I love you) / ... Oh darling I was blind to let you go...
At the end of the film, only after the death of the original Groot, did Rocket realize what he had really lost. By the time he realized who Groot really was to him, it was too late for Rocket to return or even really acknowledge that same unselfish love. Quill's sarcastic "Giving Tree" moniker from earlier in the film winds up being incredibly and hearbreakingly appropriate.