Senad Lulic tapped home the winning goal from close range in the 70th minute after Roma goalkeeper Bogdan Lobont tipped Antonio Candreva's cross into his path.
The victory means Lazio will qualify for the Europa League, salvaging an otherwise disappointing season and rubbing further salt into the wounds of their old foes, who miss out on European football despite finishing sixth one place clear of the Biancocelesti.
"I am really happy for the players who worked so hard this year and deserved the victory. It's not easy to win a game like this. We now have Europe ahead of us," Lazio manager Vladimir Petkovic told RAI Sport.
The defeat for Roma makes their manager Andrea Andreazzoli's position at the club precarious with speculation in the lead up to the match suggesting AC Milan coach Massimiliano Allegri could be a favourite to take his place.
"I don't think it's the time to be talking about my future," Andreazzoli told the post-match news conference. "It was a bad match to be honest. Neither side created much and there were a lot of errors.
"In the second half when it seemed we were in control Lazio punished us with the once chance that often decides these matches."
Local authorities now face a challenge to calm tensions between fans in the Italian capital with fears of large scale disorder following the match.
Police say they recovered two bags under a bridge close to the stadium before the match which contained bats and knives.
During a frantic opening few minutes Cristian Ledesma was booked for a clumsy foul on Marquinho before striker Miroslav Klose shot into the side netting.
With Lazio dominant for most of the first half, Klose went close again 10 minutes before the break when he forced a wonder save from Lobont with a powerful close range header.
Roma attacker Mattia Destro, who was otherwise isolated up front, nearly struck in first half stoppage time when he stretched to meet Marquinho's cross and sent his header wide of the target.
Chances were at rare in a tight and scrappy match and it was Lazio who created the first clear chance of the second half in the 67th minute when Stefano Mauri nodded down a Lulic cross into the path of the onrushing Klose, who could not make contact barely a metre from the goal.
However three minutes later Lulic opened the scoring, tapping in from close range sparking scenes of wild celebration among the Lazio fans.
Roma thought they had equalised when captain Francesco Totti's free-kick was deflected onto the bar, only for Lazio stopper Federico Marchetti to snatch the ball away from a manic goalmouth scramble.
Lazio contained Roma's late pressure with relative ease as they desperately searched for an equaliser and held out to record a famous victory.
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