Although they battled back from a half-time deficit to get a point against already-promoted Burnley, Brighton's last-gasp 2-1 win over Nottingham Forest lifted the south-coast club over Reading and into the play-off semi-finals.
Nigel Adkins' men went ahead early on when Kieran Trippier diverted a Jobi McAnuff cross into his own net but Burnley struck twice in eight minutes through Scott Arfield and Danny Ings to lead 2-1 at half-time.
Garath McCleary fired in a superb equaliser in the second half to draw Reading level, but in the end that proved not to be enough.
After relegation from the Premier League last season, Reading were hoping for a swift return. Poor home form had hampered their progress but successive wins over Middlesbrough and Doncaster had put them back in the frame.
Burnley had already clinched promotion, with two games to spare, after the 2-0 win over Wigan late last month, but that did not deter more than 2,000 Clarets fans making the long trip to the Madejski Stadium.
It was Reading who began at a furious pace and, after just 25 seconds, they made frantic appeals for a penalty after the grounded Jason Shackell appeared to inadvertently handle Pavel Pogrebnyak's goalbound shot. However, referee Mick Russell would have none of it and waved away their protests.
Reading regrouped and went ahead after 16 minutes from a fast break. Adam Le Fondre found McAnuff free on the right flank and the captain's low cross was turned into his own net from close range by Trippier.
Burnley appeared startled but responded positively and were level just four minutes later. Dean Marney was allowed space to cross from the right and, after the Reading defence had failed to clear, Arfield drilled the ball home.
It was Reading's turn to show some resolve but they were breached again in the 28th minute. Marney was again the provider, with a precise pass, and Ings made no mistake with a powerful shot for his 26th goal of the season.
Burnley began to display the style that had earned them automatic promotion and almost increased their lead on the stroke of half-time when Ings raced through alone, but he was denied by a fine save from home keeper Alex McCarthy.
With Brighton losing 1-0 at the break, Reading still had a foothold in the play-offs, and McAnuff tried to get them back on level terms soon after the interval only to blaze over when well placed.
Burnley continued to play with control, led by David Jones in midfield, but seemed fortunate when goalkeeper Tom Heaton appeared to handle a throughball from McAnuff outside his area. Again, though, referee Russell ruled in Burnley's favour.
Reading responded by equalising in the 58th minute. Chris Gunter swung over a free-kick, substitute Hal Robson-Kanu nodded down and McCleary scored with a stunning volley.
In a gripping finish, Pogrebnyak was denied by a goalline clearance from Shackell and Heaton saved well from McCleary and Alex Pearce twice. But Reading could not find the winner and were replaced by Brighton in the play-offs.