The aim of this study was to determine thegenetic variation of calpastatin gene by using PCR-RFLP method with MspI andNcoI enzymes in Kivircik lambs. Blood samples of Kivircik lambs (n=153) thatwere collected from eight different farms located in Kirklareli province, wereused for DNA isolation. After PCR amplification, products were digested withMspI and NcoI enzymes to differentiate M and N alleles on 2% and 3% agarose gelelectrophoresis. The frequency of M and N alleles were found 88.2% and 11.8%for MspI locus and 98.7% and 1.3% for NcoI locus respectively. The frequenciesof MM, MN and NN genotypes were identified 0.77, 0.22 and 0.01 respectively forMspI locus. The frequencies of MM and MN genotypes were determined 0.97 and0.03 respectively for NcoI locus, NN genotype was not observed. Observed heterozygositywas higher in MspI (0.22) than NcoI locus (0.03). MspI and NcoI loci were foundin Hardy-Weinberg equilibrium in Kivircik lambs raised in Kirklareli province.