The superstar centre hurt his calf and did not participate in Monday's field session after warm-ups in a big blow to his hopes of a NSW recall for game three in Brisbane on July 8.
Mitchell stretched and performed light drills with the team before leaving the pitch at Heffron Park with a trainer, and later going for scans.
The 29-year-old had told Fox League on Sunday night that he was "pushing" to play in round 17, pending the back injury that has sidelined him since round nine.
Souths had initially expected Mitchell would recover within a fortnight and be fit for the State of Origin series opener.
But as his recovery dragged on, Mitchell subsequently telephoned NSW coach Laurie Daley to rule himself out of game one last month.
The new injury means he's expected to miss Thursday's clash against Parramatta, with Souths fullback Jye Gray describing it as a "devastating" new setback.
"Devastating that he had to go off," Gray said after training.
"You just want to see all your teammates get out on the field and not have these injury setbacks. But he we will be back.
"We're just hopeful, obviously we don't know how bad anything is at the moment. Obviously devastated for him."
After their game two humiliation, NSW would have been hopeful of calling on the talismanic Mitchell, who has played 11 Origin games and is usually a walk-up starter when fit.
Kotoni Staggs had an especially tough night in Origin II, while the Blues' other incumbent centre Tolu Koula has played only two games at Origin level.
It remains to be seen whether the Blues would risk picking Mitchell if he recovers in the coming days, given his last game was on May 3.
"I think he probably goes back into that side if he was fit," said Souths forward Lachlan Hubner.
"But as a Queenslander, happy days (if he is out)."
There are a handful of leading options to come into the team if Blues selectors wish to make a change in the centres but opt against taking a chance on Mitchell.
Canterbury's Stephen Crichton returned from a shoulder problem to kick the game-winning field goal against Manly on Saturday night.
Bradman Best set up a try on his return from a calf injury in Newcastle's defeat of St George Illawarra and has been excellent in two previous Origin games.
Ruled out of Origin II during camp, Penrith centre Casey McLean is considered unlikely to recover in time for this weekend's clash with North Queensland.
But the 20-year-old could have shaken a quad injury in time for the decider if the Blues opt to recall him into their squad.
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