The aim of this study was to determine the genetic variation of calpastatin gene by using PCR-RFLP method with Mspl and Ncol enzymes in Kivircik lambs. Blood samples of Kivircik lambs (n=153) that were collected from eight different farms located in Kirklareli province, were used for DNA isolation. After PCR amplification, products were digested with Mspl and Ncol enzymes to differentiate M and N alleles on 2% and 3% agarose gel electrophoresis. The frequency of M and N alleles were found 88.2% and 11.8% for Mspl locus and 98.7% and 1.3% for Ncol locus respectively. The frequencies of MM, MN and NN genotypes were identified 0.77, 0.22 and 0.01 respectively for Mspl locus. The frequencies of MM and MN genotypes were determined 0.97 and 0.03 respectively for Ncol locus, NN genotype was not observed. Observed heterozygosity was higher in Mspl (0.22) than Ncol locus (0.03). Mspl and Ncol loci were found in Hardy-Weinberg equilibrium in Kivircik lambs raised in Kirklareli province.