Halfpenny linked up with the squad in Johannesburg last week, but he has now aggravated the same injury in training.
He was to have been on the bench for tomorrow night's fourth tour fixture against the Sharks at Absa Stadium, but his place has now been taken among the substitutes by Wales colleague James Hook.
Halfpenny will undergo a scan to try and gauge the full extent of the injury, and the Lions are currently waiting for an update.
"He recovered very well from the last time," said Lions assistant coach Shaun Edwards.
"He was doing some extra work at the end of training yesterday and he just felt it strain."
Halfpenny burst onto the international scene last autumn, but this latest setback will be a concern for both the player and Lions management.
The Lions have so far reeled off successive victories against a Royal XV, Golden Lions and Cheetahs as the build-up continues towards next week's first Test against world champions South Africa in Durban.