The South Sydney talisman has been sidelined since round nine with a back issue, but returned to training over the weekend, sparking hopes he'd be fit for the Rabbitohs' match against Parramatta on Thursday night, and for Origin III.
But Souths coach Wayne Bennett on Tuesday said Mitchell's new calf injury - stemming from his back ailment - had put the 29-year-old out of both games at least.
"He's done his calf muscle," Bennett told reporters.
"It's coming from his lower back. It will be a couple of weeks. He was warming up this morning and he felt a bit of soreness there.
"No-one is to blame. He's definitely out of Origin."
After feeling his calf during warm-ups at Souths' training session on Monday, Mitchell stretched and performed light drills with the team before leaving the pitch at Heffron Park with a trainer, before going for scans.
The superstar centre had told Fox League on Sunday night that he was "pushing" to play in round 17, pending recovery from the back injury.
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.
Preparing to face Parramatta without Mitchell, Souths fullback Jye Gray described his teammate's injury as a major 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 will be back.
"Obviously devastated for him."
After their game two humiliation, NSW would have been hopeful of calling on 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 for NSW.
"I think he probably goes back into that (NSW) side if he was fit," said Souths forward Lachlan Hubner.
"But as a Queenslander, happy days (if he is out)."
A handful of leading options remain if Blues selectors wish to make a change in the centres.
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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