Several characters, with varying degrees of harshness, tell Hiccup that Toothless has the Light Fury now and no need for him. The saddest thing about this is that they're right. Toothless, after he receives his new tail fin, flies off with the Light Fury and does not come back until prompted by Hiccup. Ultimately, they choose to take the flock away to the Hidden World for their safety, with Hiccup and Toothless parting ways for good.
While he and Astrid watch Toothless and the Light Fury's coronation and mating dance, you can just see the moment Hiccup realizes how right those same people were about Toothless not needing him anymore after Astrid's awed remark. His happiness and support for Toothless finding the Light Fury was genuine, yes, but it's only now sunk in for him that he really will have to say goodbye.
Leading off of this realization, Hiccup gets an even nastier answer to his ideals of peaceful human-dragon co-existence when he and Astrid are caught by one of the wild dragons watching the procession, prompting Toothless to fly off and save them both. With the silence that follows, what should have been a joyful reunion and ride back to New Berk is tainted by two more of Hiccup's greatest hopes being dashed: after the attack, it's clear to him and Astrid that the Berkians cannot live within the Hidden World with their dragons, and after putting his support behind Toothless and the Light Fury becoming a couple, Hiccup just forcefully (if unintentionally) separated them both shortly after they had officiated their bond with each other.
Grimmel makes two emotional jabs at Hiccup when they encounter each other: one similar to Drago's that touched on his father ("What shame [Stoick] must feel [about someone like Hiccup being the Dragon Master]"/ "[Stoick changing his mind about dragons] was the last mistake he ever made"), and another that touched on Hiccup's dependency on Toothless when he captures Toothless and the Light Fury, and forces Toothless to choose between her and Berk's dragons ("Face it— you're nothing without your dragon!"). Hiccup's essentially having his greatest failure and his greatest weakness thrown in his face; but he's had a year to come to terms with Stoick's death, and years longer to get over his insecurities about his self-worth and his place in the world. The latter, however, actually hits him where it hurts; Hiccup's already trying to acknowledge that he's losing Toothless after years of being inseparable with him, but some of his actions as chief to keep human-dragon co-existence and his friendship with Toothless left the Berkians, his friends, and his family defenseless against an enemy. No wonder he's left feeling like the same failure from the first film.
Hiccup telling the Light Fury to go save an unconscious Toothless after he pulls off her poison saddle and lets her regain lucidity. After he lets go, it looks like he's losing the mid-air assault with Grimmel before she does come back to catch him.
Not just losing— though he does start fighting back, it at first doesn't look like Hiccup even puts up much of a fight against Grimmel as they're both falling, even when he gets caught off guard at the latter tearing off his wings. It looks more like he's sincerely bracing himself for his death.
The whole moment where the people of Berk let their dragons go, starting with Hiccup reversing his touch on Toothless' nose. There is not a dry eye among any of them. As they all fly away, Toothless is the last to go, sharing one last lingering look with Hiccup.
The perfect reversal shot deserves its own bullet point—it is utterly devastating. All of the overwhelming emotion of that first iconic touch, the softness, the gentle pink lighting—even down to the momentary, terrified beat of hesitation. Only this is the last touch they'll ever share, and it's clear that both of them know it. The way Hiccup's fingers flinch back toward Toothless, just for a moment...
To those who have watched Dragons: Riders of Berk, it's quite likely that among the flock of dragons heading back to the Hidden World includes fan-favorites such as Torch, Thornado, Windshear, Shattermaster, etc.
Hiccup is missing his prosthetic leg during the entire scene. While it might look silly with him forced to hop around during such a dramatic moment, it really drives home the point that Hiccup is incomplete without his dragon. And yet he tries to stand tall and proud as well as he can.
Among the Vikings who have to say goodbye to their dragons, one particularly sad example would be Fishlegs, especially seeing the doting amount of love he has for his Gronckle Meatlug as we see in the series. It's worse when you remember how much of a dragon nerd he is: he gets so thrilled every time he sees a rare or new species. And now he'll never see them again.
It's also unclear whether the rest of the Viking teens (and, if we're going by the series, Berk's former Auxiliary team/ the Berserkers/ Outcast tribe members/ Defenders of the Wing/ Wingmaidens) are ever reunited with their dragons.
Gothi the Elder saying goodbye to her little flock of Terrible Terrors is particularly sad, as she was already old when Hiccup made peace with the dragons, and despite having lived her whole life fearing them, she came to have a change of heart and accepted a small bunch of them as her beloved pets. And given her advanced age, it's unlikely she'll live long enough to see them return.
The epilogue. Judging by the ages of Hiccup and Astrid's two children, and Hiccup's own bearded face and small wrinkles by his eyes, it appears to skip at least a decade before he travels with his family to visit Toothless. As they come across him with the Light Fury and children of their own, Toothless doesn't recognize Hiccup and jumps onto their boat with a menacing glare, meaning to scare away the humans coming too close to his mate and offspring. Hiccup carefully reaches his hand out and asks with tears in his eyes, "Remember me, bud?", and as Toothless gets a whiff of his scent, finally recognizes his old best friend and is happy beyond words, tackling Hiccup to the boat's floor and covering him with licks while Hiccup and Astrid's children watch in awe. Hiccup then introduces his children and Toothless to each other, and the children greet Toothless by reaching out their hands to touch his snout, Hiccup watching with great joy as they do.
And the sad fact that Stoick never lived to see his grandchildren.
Toothless being unable to fly as he tries to catch up with the Light Fury, twice. His sorrowful moans of calling out to her, almost begging for her to come back, is very heartbreaking to listen to.
And the way he sadly watches two Nadders courting. He's all alone and lonely, even with Hiccup, and the reveal that he really is the last Night Fury makes it all the worse.
During Hiccup's Heroic BSoD, Valka and Astrid have a poignant conversation concerning the former's observation that Hiccup thinks he has to lead by himself— because that's what Stoick was forced to do. There's definitely a part of Valka that constantly regrets hurting her family by staying away from Berk, even as she's re-adjusting to living there in its changed dragon-loving environment again.
Heartwarmingly enough, that doesn't have to be the case with Hiccup, and from that point on it's clear that he and Astrid are going to be inseparable: they find the Hidden World together, they rescue the dragons from Grimmel together, they rule over the tribe together, and over a decade later, with their two children, they reunite with their dragons together.
When Hiccup approaches Toothless at the end of the movie, Toothless boards the boat without recognizing him and appears on the verge of attacking him before catching his scent. Word of God confirms that Toothless did, indeed, forget about Hiccup for a little bit due to his adult appearance, but eventually realized it was him after catching his scent.
Dean DeBlois: Ten years had passed, and [Toothless] had kind of forgotten his former life. It takes him a moment, especially with Hiccup looking different.
In the same interview, DeBlois saying that the reunion was meant to be a "final joy ride", but not an indication that Toothless and Hiccup ever see each other again after the reunion.
The flashbacks where Stoick tells Hiccup about the Hidden World. In the light of HTTYD 2, just seeing how caring he was and that he only wanted a peaceful life for his son and his village. Even more touching for those who watched Hiccup and Stoick bond in the TV and Netflix series.
The fact that Berkians and dragons were bitter enemies for seven generations and friends for only 5 years before the dragons left.
After the dragons leave, Fishlegs can be seen carrying around a doll of Fishmeat.
The fact that everything that was ever lost in the name of defending dragon-human unity is rendered meaningless. Hiccup's lost leg, Valka's 20 years away from her family, Stoick's sacrifice... ultimately, it was all for nothing.
Steven Spielberg himself has read the film's script, and was almost unable to make it to the end due to his tears.
Word of God revealing that the Light Fury is a distinct variant of Fury and not an actual Night Fury — and that "unlike Toothless", she's not the last of her kind. In other words, the species is gone forever, and Hiccup and Toothless's dreams of someday finding other Night Furies never do come true. They do have offspring together in the end, but without other Night Furies to mate with, this trait will quickly be bred out of them. Essentially, it's like the world's last white tiger falling in love with a lion...