The South Sydney talisman suffered the fresh injury at training on Monday, just as he had been hoping to return from the back issue that had sidelined him since round nine.
Scans confirmed a calf tear that will rule Mitchell out of contention for Origin III at Suncorp Stadium on July 8, and extend his time on the sidelines by at least a few weeks.
Mitchell had been hopeful of facing Parramatta on Thursday night in the Rabbitohs' last game before teams are picked for Origin III.
But his unavailability is another blow for a Blues team fighting to recover from a 44-24 humiliation in the second game of the series.
A veteran of 11 Origin games, Mitchell has usually been considered a walk-up starter for a spot in the centres 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.
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.
Mitchell's injury continues a frustrating season for the 29-year-old, who had initially been expected to recover from his back issue inside a fortnight and be fit for Origin I.
But as his recovery from that injury dragged on, Mitchell telephoned NSW coach Laurie Daley to rule himself out of last month's series opener.
He has still not returned to the field since then.
Preparing to face Parramatta without Mitchell, Souths fullback Jye Gray described his teammate's latest 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.Â
"Obviously devastated for him."
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