On-loan midfielder Rohan Ricketts shattered Reading's play-off dream with a late winner to sink Glenn Hoddle's home-town club.
Hoddle, who lives just down the road from the Madejski Stadium, saw his side fight back from a goal down at half time to extend their remarkable unbeaten record to 17 games without defeat.
Nicky Forster put the Royals ahead after only eight minutes, but the visitors hit back to level through Leon Clarke before Ricketts snatched victory late on.
Defeat saw Reading slip back out of the play-off places as West Ham now head them on goals scored, meaning Reading need to better West Ham's result at Watford when the Royals travel to Wigan on the final day of the season.
It was the second time in three seasons that Wolves have wrecked Reading's play-off hopes, having beaten the Royals in the semi-final on their way to promotion in 2003.
But defeat had looked an unlikely scenario after Reading got off to just the start they needed courtesy of Forster's precise volley.
Dave Kitson rose well to flick on Marcus Hahnemann's long punt forward and Forster pounced to hit a sweet volley beyond the reach of Wolves keeper Michael Oakes.
Victory would have been enough to take Reading three points clear of seventh-placed West Ham with only one game of the season remaining.
And Steve Coppell's side appeared content to protect their narrow advantage as the visitors created the better of the chances.
South Korean midfielder Ki-Hyeon Seol saw a shot flash wide and playmaker Seyi Olofinjana sent a 30-yard drive just the wrong side of a post.
Top-scorer Kenny Miller was a threat too, but the best of his chances flew straight at Reading keeper Hahnemann.
But the big American keeper was beaten eight minutes into the second half as Wolves deservedly got back on level terms.
Andy Hughes conceded an unnecessary corner when he headed behind under little pressure and Wolves were quick to cash in as Clarke beat Martin Keown at the near post to head home Lee Naylor's corner.
Reading failed to respond and Olofinjana almost put the visitors ahead with a volley from the edge of the box that dipped just over.
Ricketts then threatened with an angled drive that flew across goal, but the same player snatched victory for Hoddle's side when he pounced on a mistake by Ibrahim Sonko and eased past the same defender before side-footing beyond Hahnemann.
Substitute Les Ferdinand was agonisingly close to an injury-time equaliser for the Royals, but Oakes grab the ball at the second attempt after an initial fumble.