Calpastatin (CAST) is an endogenous and specific inhibitor of calpains found in meat. Because of this feature, CAST is a major gene that directly changes the toughness of meat. The objective of this study to determine the CAST gene polymorphisms in Kıvırcık crossbred ewes. Therefore, CAST gene polymorphisms were investigated in 100 Kıvırcık crossbred ewes grown in Thrace region. The PCR-RFLP method was used to determine the genetic variations of the CAST gene. In the current study, the estimated frequencies of three genotypes including MM, MN and NN at CAST/MspI polymorphism were 0.82, 0.16 and 0.02 and they were 0.90 and 0.10 for M and N alleles, respectively. There was no deviation from Hardy-Weinberg equilibrium (P>0.05) relative to CAST genotypes.