Initially named 18th man, Penrith's Stephen Crichton will replace Mitchell at left centre at Suncorp Stadium on June 21, having already done so in NSW's Game I loss last month.
Canterbury co-captain Matt Burton was promoted to 18th man for the series opener and will join camp to reprise the role for Game II.
Mitchell's South Sydney teammate Keaon Koloamatangi remains 19th man.
Mitchell tweaked his calf during the Blues' camp for Game I and has not played since, but resumed running at training over the long weekend so was named on Brad Fittler's team sheet on Tuesday.
But Souths coach Jason Demetriou told AAP Mitchell still had hurdles to clear before he could be approved to play in the must-win second Origin game.
Fittler conceded it had been a "risk" naming Mitchell as well as Cam Murray and Liam Martin, who are under injury clouds of their own but are tracking to play.
Mitchell missed NSW's whole-squad media day on Wednesday, instead consulting with the Blues' physiotherapists.
His teammates shrugged off that absence at the time, but the following afternoon, on the first rest day of the Blues' camp, it was confirmed the Rabbitohs star would miss the second Origin game.
"The decision was made by Westpac NSW Blues coach Brad Fittler, Mitchell and the medical staff after they determined he would not have overcome the injury in time for the game at Suncorp Stadium in Brisbane on Wednesday 21 June," an NSWRL statement read.
NSW would have been counting on Mitchell, one of the most explosive players in the NRL, to help spark their attack in Brisbane after they faltered in Game I.
Instead, he sits out a fifth consecutive Origin match. Mitchell missed the first two games of last year's series with a hamstring injury, then withdrew from contention for the third to focus on club football.Â
Filling in for Mitchell, Crichton was one of NSW's best in the series opener, scoring the try that gave them the lead for the first time in the final 15 minutes.
He has already been training in Mitchell's place in the first two days of camp and will do so again when the Blues return to Coogee Oval on Friday morning.
"As 18th man, I'll just be ready for anything," Crichton told AAP on Wednesday.
"Basically it's just anything for the team."
Wednesday's match will be Crichton's fifth for NSW.
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