Benzema's injury is the latest in a long list of injury absentees for France, who have also lost the services of Paul Pogba, N'golo Kante, Presnel Kimpembe and Christopher Nkunku in the build up to the tournament.
Benzema won the prestigious Ballon D'or award last month but has been dogged by fitness issues this term for club side Real Madrid.
The 34-year-old played just 28 minutes in Real Madrid's last six matches before the break for the World Cup as he was troubled by muscle fatigue.
Benzema was involved in full training for the French side for the first time on Saturday but was unable to complete the session after suffering a thigh injury.
The French Football Federation said in a statement: "Karim Benzema has pulled out of the World Cup with a thigh injury. The whole team shares Karim's disappointment and wishes him a speedy recovery."
It is estimated that Benzema's injury will keep him out of action for three weeks and France have until Monday to call up a replacement.
France manager Didier Deschamps said: "I'm extremely sorry for Karim who had made this World Cup a major objective."
"Despite this new blow, I have every confidence in my squad. We are going to do everything to meet the immense challenge that awaits us."
France start their defence of the title they won four years ago in Russia against Australia on Tuesday before further matches with Denmark and Tunisia.
