The Manchester United forward had been expected to start alongside club team-mate Wayne Rooney and Daniel Sturridge when England get their Group D campaign under way in Manaus, but Welbeck's place now looks in doubt.
Welbeck, 23, picked up the knock in training on Tuesday and failed to make an appearance at the beginning of Wednesday's session.
Welbeck's position as a starter for England was called into question during the team's pre-tournament friendlies against Peru, Ecuador and Honduras, producing unspectacular performances in each game.
Should the United youth product not prove his fitness in time for the meeting with Cesare Prandelli's side, Liverpool winger Raheem Sterling may deputise.
Welbeck scored four goals in eight appearances in qualifying and he joins Alex Oxlade-Chamberlain as an injury worry, with Roy Hodgson hopeful that the Arsenal midfielder will be ready to return to action on June 19 against Uruguay.
