He limped out of the Ospreys' European Cup defeat to Stade Francais in Paris on Friday night and is set for a scan to assess the extent of the problem.
"Jonathan has suffered the same hamstring injury that has troubled him previously. It doesn't allow him to run over 50 per cent," said Ospreys coach Lyn Jones.
"Is he a doubt for Twickenham? He has come off injured with two weeks to go to the England match. Make your own assumptions."
It is the latest fitness headache to be put in front of Ruddock, who is already without injured quartet Ryan Jones, Brent Cockbain, Tom Shanklin and Kevin Morgan.
He is also minus Gavin Henson, who is unavailable for the first two matches while he serves out a seven-week ban.
Ruddock also has real concerns over his Grand Slam captain Gareth Thomas, who played for Toulouse against Llanelli Scarlets on Saturday with very heavy strapping on his troublesome right knee.
Thomas remains upbeat about the injury and is planning to play in Toulouse's French Championship clash against Pau on Saturday.