Watford missed out on automatic promotion after losing to Leeds in a game delayed by serious injury to keeper Jonathan Bond.
Dominic Poleon put Leeds in front, but Almen Abdi levelled shortly after.
Watford's Troy Deeney then saw red for two bookable offences before news of a late Cardiff equaliser at Hull left the Hornets needing a goal to go up.
But Ross McCormack scored Leeds' winner with the help of a blunder by replacement keeper Jack Bonham.
Watford will now turn to a play-off semi-final against Leicester City in pursuit of promotion and will hope to recover from an emotionally draining day to replicate their play-off victories in 1999 and 2006.
The Hornets have been out of the top flight for six years now, but have been genuine promotion contenders all season with the dawning of a new era under owners the Pozzo family.
The Italians have injected life into the Hertfordshire club, firstly by appointing Gianfranco Zola as boss and then by bringing in players on loan from Udinese and Granada - both clubs also owned by the family.
It has been a controversial approach, with nine members of the current squad belonging to either the Italian or Spanish sides, but it has unquestionably been effective.
Both Watford and Leeds were unsettled by late changes before kick-off, with Hornets keeper Manuel Almunia replaced by Bond and United midfielder Zac Thompson making way for Michael Brown.
And the changes continued early on, with Leeds' Steve Morison taking a blow to the head and being replaced by Poleon, who was at close quarters when Bond collided with his own defender and was taken off on a stretcher to make way for the entrance of 19-year-old academy product Bonham.
It was Poleon who would then get the goal to put his side in front, rolling into an empty net after Bonham and Joel Ekstrand got into a muddle trying to deal with Tom Lees's long ball.
Watford's deflation was short lived however when Abdi collected the ball from Ikechi Anya, who had surged in off the touchline, and curled a delightful finish beyond Paddy Kenny from 20 yards out.
Brian McDermott, who was winning promotion with Reading this time last year, will be pleased his mid-table Leeds side arrived with the commitment and desire to play their part in the final day permutations.
His men became withdrawn late on though, particularly after Deeney's second yellow card for a challenge on Michael Brown.
With the game heavily delayed by the earlier injuries, news of Cardiff's equaliser at Hull filtered through the crowd and the Hornets, urged on by a wave of noise, hunted the goal that would put them into the Premier League, while the Tigers could only sit back and watch.
Kenny made a fine double save from Abdi's shot and Nathaniel Chalobah's follow-up to kept the home side at bay.
And as Zola's men threw everything forward, Leeds finished off their promotion dreams when Aiden White broke clear and fed McCormack, who delivered a lob that debutant Bonham failed to stop, despite getting a good hand to it.
McDermott will look to mould his own promotion charge next season off the back of this final day flourish, while for Watford that ambition is still very much a possibility this term, but they will have to do it the hard way.
Watford boss Gianfranco Zola:
"I was shouting to my players, encouraging them, but they had given everything. I cannot complain.
"Manuel Almunia had a problem in his left hamstring. That's why we had to go with Jonathan Bond.
"With Jonathan the first impression is he has a broken nose. But he's been taken to hospital for assessment.
"It looks like it's not too bad for Almunia and we're hopeful he can be back [to face Leicester in Thursday] as we know Leicester will be a tough game."
Leeds boss Brian McDermott:
"It was an amazing game. We kind of knew what was going on at Hull, but we did what we had to do.
"We were spot on and did our business for our club. Hopefully Jonathan Bond is OK. Steve Morison has concussion.
"Dominic Poleon is a young boy from the academy with real pace and power. We need to get him really fit, if we do that this summer he could be a real asset to us.
"Gianfranco is a friend of mine. I hope Watford do well as Gianfranco is a top person, he's done a fantastic job and they have a chance in the play-offs."