Pompey's promotion bid suffered a setback when fellow hopefuls Doncaster took all three points at Fratton Park.
The Blues gifted Rovers a 2-0 goal and despite a gallant fightback they could not salvage a draw.
Matt Clarke came into the defence in place of Tom Davies but that was the only change - but Paul Cook might have wished he had made more as Doncaster showed their credentials by going 2-0 up.
They were ahead on seven minutes when Pompey lost possession too easily in midfield and a long ball over the top caught out the defence. John Marquis chased it and seized on hesitancy to take it round David Forde and slot home.
Andy Williams doubled Doncaster's lead 11 minutes before half-time when he received a free-kick with his back to goal, turned and fired in past Forde.
The home crowd were stunned but Pompey did at least start to show some fight after Rovers' second.
Gareth Evans was denied a penalty when ref Darren Deadman ruled that he had dived, but Carl Baker rifled in to make it 2-1 after a defender made a mess of clearing an Evans cross.
Pompey had the lion's share of the play in the second half but not even the introduction of Milan Lalkovic, Amine Linganzi and Curtis Main could bring about an equaliser.
Baker, Main and Roberts all went agonisingly close to making it 2-2 but it wasn't to be and the Blues slipped to sixth place.
Cook told the club website: 'It was always going to be a tough game against a very good side, but we certainly didn’t start well enough. We found ourselves two goals down — and deservedly so. We tried to get back up and scrap, but never really found any fluidity.'
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