The Newcastle forward injured his thigh during England's 1-0 friendly victory over Austria on Friday, and manager Steve McClaren was left to bemoan another setback.
"He won't be fit for Wednesday, It was a strange one, it was his standing foot. He's pulled his thigh and unfortunately he's out," McClaren told a post-match conference.
"That's football but we do seem to be plagued with injuries at the present moment and for what I believe will be our biggest game for quite some while we're going in without two centre halves and two centre forwards who we would pick."
Owen joins regular forward Wayne Rooney on the sidelines ahead of the must-win Group E clash, as well as key defenders John Terry and Rio Ferdinand.
Owen was replaced by Tottenham Hotspur forward Jermaine Defoe for the Croatia game.
England lie second on 23 points in a very tight Group E, three points behind leaders Croatia and two points ahead of Russia, but the latter have a game in hand.