Wolfe was helped onto a cart, driven off the practice field, and helped into the trainer's room while putting no weight on his right leg.
There have been other injuries on the defense the past couple months, as well.
His backup, Shaq Barrett, will miss an extended period of time due to a hip injury.
The Broncos defensive line was already thinning when second-string nose tackle Billy Winn went down with what turned out to be a season-ending ACL tear during Thursday's preseason opener against Chicago.
Wolfe was in a pile during a running play in a team drill early in practice - tackle Menelik Watson limped, flexing his own right leg following the play - and stayed down. He missed all of last season with a knee injury suffered in training camp last season while going against Russell Okung. Wolfe posted 5.5 sacks each of the past two years.
The Broncos added free agent rookie Nelson Adams for depth on Saturday. The team could also consider moving second-round rookie DeMarcus Walker back to defensive end, although head coach Vance Joseph said he could him at outside linebacker for now. Losing Wolfe, their best defensive lineman, would have struck a major blow to a defense looking to improve significantly against the run.