The Manchester City midfielder was replaced at half-time during Friday's first leg due to a tight hamstring and, after being assessed by the England medical team, it was announced he would be returning to his club.
Rodwell will therefore miss Tuesday's return leg against Serbia in Krusevac, where England will defend a 1-0 lead as they bid to reach next summer's finals.
Coach Stuart Pearce said: "We're disappointed for Jack, naturally, but I know what I've got in this squad and I know they're good enough to see the job through.
"We've had a lot of injuries to contend with throughout this qualification campaign so it's not a challenge we're unfamiliar with. I know my players and I'm no less confident in our chances."
Pearce, who is also without Liverpool defender Jack Robinson and Norwich goalkeeper Declan Rudd, has decided against calling up any replacements for the match.
Meanwhile, Pearce says he is confident his side will not be su bjected to racist abuse at the Mladost Stadium in Krusevac.
Dealing with the Serbian threat on the field is the priority, with it hoped that those in the stands at the 10,330-capacity venue will not steal the headlines for all of the wrong reasons.
Pearce believes there will be no repeat of the scenes which marred England's last meeting with Serbia, at the 2007 championships, when UEFA officials were forced into an impromptu anti-racism announcement after Nedum Onuoha was targeted by a pocket of supporters.
"That was some years ago and we have no fears in that respect," said Pearce.
"I believe that everything has moved on since then. My only concern is the match and making sure our players are prepared for the game and any eventuality that may occur during it.
"Anything else over and above that will be dealt with by the authorities and the referee, as it normally is on the night."
England head into the midweek tie looking to secure t heir safe passage to a fourth straight Euro finals, and Pearce is confident they can get the job done.
He added: "We have got some experienced heads in the dressing room. I think they are mature enough to deal with the atmosphere they will face.
"Admittedly, they are only young but every time we have taken them into Europe, we think they have had a decent grounding at their club.
"This tie is far from finished and they'll make it very unpleasant for us but we will set the team up to win."
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