Marker assistant selection is one of the best methods to improve the accuracy of prediction and response to selection. In studies related to weight gain and meat quality Calpastatin gene has been identified as a candidate gene. This gene is located on cattle chromosome 7 and is effective on formation, lysis of muscle texture and meat tenderness after slaughtering animals. In order to study polymorphism of Calpastatin gene in Holstein cattle 50 animals from Behin Taliseh corporation were randomly sampled. DNA extraction was carried out by modified salting out method. Polymerase chain reaction was performed to amplify a 622bp fragment of the L region of exon and entron 1 of this gene. Restriction reaction of PCR products was done using MspI enzyme. Then all the animals were genotyped base on digested fragment profile, respectively. Frequencies of MM, MN and NN genotypes were 46%, 54% and 0% for Holstein cattle and 0%, 45% and 55% for Buffalo. Frequencies of M and N alleles were 0.73 and 0.27, respectively for Holstein cattle and 0.225 and 0.775 for Buffalo. Results showed that this locus was not at Hardy–Weinberg equilibrium in the Holstein cattle (P<0.05) while Buffalo herd showed equilibrium.