Barkley has been ruled out of the clash with Wales at Twickenham on February 4 after sustaining a dislocated right thumb during Bath's 35-23 European Cup defeat by Leinster on Sunday.
The 24-year-old's right hand is in a plaster cast and he will visit a specialist to assess the extent of the damage.
England head coach Andy Robinson said: "It's unfortunate for Olly, Bath and England because he's an important part of the squad.
"At this stage it's too early to say how long he will be out of action but we wish him all the best and a speedy recovery."
Barkley had been widely tipped to start the Wales game at inside centre with the explosive Josh Lewsey alongside him in a dynamic new midfield.
England's alarming lack of creativity behind the scrum has been exposed in recent seasons and the Barkley-Lewsey combination was seen as a possible solution.
Barkley, who is left-footed, would also have provided a useful foil to Sale fly-half Charlie Hodgson's right boot and the 14-cap international is a solid performer with the vision to unlock defences.
Robinson still has plenty of cover available at inside centre with Mike Tindall, Stuart Abbott and Jamie Noon in his Six Nations squad but Barkley's loss will be keenly felt, not least because he can provide cover at centre and fly-half.
The injury has also ruled him out of Saturday's Guinness Premiership clash with Wasps at the Recreation Ground.
Bath head coach Brian Ashton said: "It's desperate bad luck for someone who is in form at the moment.
"Olly would probably have made the England 22 to face Wales if not the starting XV. We would like to wish him a rapid recovery."
Barkley's hopes of beginning England's RBS 6 Nations campaign may be over but Wasps number eight Lawrence Dallaglio is desperate to complete his dramatic comeback for the world champions.
Dallaglio, who amassed 73 caps before announcing his international retirement 17 months ago, returned to England training at Loughborough on Tuesday.
The 33-year-old admits he is revelling in his return but is under no illusions he will be an automatic selection for the opener against Wales.
He said: "I'm delighted to be back. It's a first step for me - I've been asking for it, it's happened and now I'm back in the squad.
"But I know as well as anyone the amount of hard work it's going to take to get back in the team and then to stay in it.
"I said from the outset that it's important to have competition for places in the squad and if we can have that in as many positions then we are going to get stronger and stronger.
"The Six Nations is a tough tournament to play in and from my previous experience, when we won the Grand Slam in 2003, I think we used 36 players across the five games.
"It is going to be a squad effort and the quality players here are a reflection of what is going on in the game at the moment.
"I don't know when my chance will come - it may come in the first game, it may not, but I hope at some stage it will come and it's a question of being in the right shape physically and mentally to take the opportunity when it does.
"England have moved on, and rightly so, from that whole World Cup experience and this is very much a different squad, all be it with the same target to win every match."