Benzema was substituted after 42 minutes of the game at Estadio de Vallecas, with Lucas Vazquez coming on in his place. Vazquez subsequently scored as Real came from 2-0 down to claim three crucial points with a 3-2 success.
The Frenchman appeared to sustain a knock to his right knee, which will concern Madrid supporters ahead of the first leg of their European semi-final at the Etihad Stadium next week.
Real are already sweating over the fitness of Cristiano Ronaldo for the game against the Premier League side.
The Portuguese international was ruled out of the meeting with Vallecano after suffering a muscle injury in the 3-0 victory over Villarreal on Wednesday and is a doubt for the City tie.
He fell awkwardly and was forced to come off moments before the final whistle.