Harry Kane missed England's final World Cup warm-up match at home on Tuesday after suffering a "minor issue in training".
The captain, who only joined the squad at the weekend after being rested for the first half of the international camp, has enjoyed a spectacular season for Bayern Munich, scoring 48 goals in 40 matches.
"Harry Kane is rested tonight as a precaution having picked up a minor issue in training, but remains with the squad receiving further assessment," the Football Association said in a statement on X.
England boss Thomas Tuchel, speaking ahead of kick-off at Wembley, said the injury came "from basically out of nothing".
"It just happened," he told ITV. "He had to step out of training yesterday after 15 minutes. No chance to be involved, not even today.
"He is being further assessed now and will be in the stadium to watch. It is serious enough to not be able to play."
Kane, 32, is England's all-time record goalscorer, with 78 goals.