The Spain international left the field in tears, with his prospects of playing in the Champions League final now in severe doubtDiego Costa limped off during Barcelona and Atletico Madrid's title decider at Camp Nou on Saturday after suffering a recurrence of a hamstring injury.
The Spain international has battled with the problem in recent weeks but was passed fit for the clash by coach Diego Simeone.
However, Costa's afternoon lasted just 16 minutes, with the striker bursting into tears on the side of the pitch following his withdrawal.
The problem now casts severe doubt over his involvement in the Champions League final against Real Madrid next week.
Simeone's side could also be without Arda Turan, who was withdrawn just minutes later and replaced by Raul Garcia following a knock to the hip.
Atletico need to avoid defeat against Barcelona to lift La Liga for the first time since 1996.