Abstract: Data on genetic differences at CSN3 (κ-casein) in bovine breeds including a DNA based typing method and the mutation responsible for variation on DNA level using the Polymerase Chain Reaction (PCR) followed by restriction enzyme digestion (to reveal a RFLP) of blood samples was collected from indigenous Iranian cattle breed Sarabi and Holstein-Friesian. The PCR products were digested with Hind III and then loaded onto agarose gels, stained with ethidium bromide. Gene frequencies for A and B alleles in holstein and sarabi cattle breeds were estimated 0.8284, 0.1716 and 0.7652, 0.2348, respectively. Comparison of allele frequencies indicated that there was no significant difference between observed and expected frequencies in both Sarabi and Holstein populations. The observed hetrozygosity for Sarabi and Holstein were 0.3484 and 0.2843, respectively .The estimated gene diversity from sum of squares of allele frequency, for Sarabi and Holstein were 0.3593 and 0.2843, respectively. Based on information at this locus, no evidence was found for disequilibrium in two populations. Comparison of allele frequencies of this study with those of other cattle breeds indicates that only significant differences was demonstrated between two breeds of our study with Jersey.