The gap between leaders Doncaster and fourth-placed Pompey has increased to 10 points after Rovers inflicted a 3-1 defeat on Paul Cook's side at the Keepmoat.
A double from former Blues striker John Marquis started and finished the scoring either side of Kal Naismith's equaliser and Tommy Rowe's neat finish.
It was a big disappointment for the Blues after they had won three and drawn two of their previous five games, but there were encouraging signs too from a Blues side who did more than their fair share of attacking, just without the killer touch in the box.
Doncaster took just five minutes to take the lead and it was Marquis who buried it after James Coppinger's ball in was flicked on by Matty Blair.
Pompey had started slugglishly and the worry was that Rovers would be two up before Cook's men had even begun to play. Gradually Pompey did come into the game more and home keeper Marko Marosi made one splendid one-handed save low down to his right to keep out a close-range header.
Pompey got better as the first half wore on and four minutes before the break Naismith finished coolly after Kyle Bennett had intercepted a loose pass and played him in on goal.
That left the 600-plus Pompey fans who made the unappetising trip north on a freezing Thursday thinking their team had the momentum - but they could not build on it in the second half.
On 58 minutes Doncaster retook the lead when Coppinger ran at the Blues defence and fed Rowe, who overcame a half challenge from Gareth Evans before rifling a shot high into David Forde's top corner.
Milan Lalkovic and Gary Roberts were brought on but before they'd had a chance to influence a fightback Rovers were 3-1 up as Marquis' superb finish was applied to a cross from the left by Rowe.
It was hard to see how Pompey could get back into it from here - they'd need a comeback like the one at the same ground in 2012 which saw them turn a late 3-2 deficit into a 4-3 win. This time there were no such heroics, though they had plenty of the possession in the final 15 minutes.
Lalkovic had a header well parried away by Marosi, who was then stretchered off with a nasty-looking ankle injury, and Michael Smith sent one header from a right-sided cross well over the bar.
But Rovers would not be breached and held on for a win that extends their lead and puts the pressure back on Pompey to find another unbeaten run from somewhere to reignite their hopes of finishing in the top three.
Pompey (4-2-3-1): Forde; Evans, Burgess, Clarke, Stevens; Rose, Doyle (c); Baker (Lalkovic 62), Naismith (Chaplin 75), Bennett (Roberts 67); Smith. Subs not used: O’Brien, Whatmough, Linganzi, Hunt
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