Nutrient compositions of Milk are suitable and perfect components among foods that is consumed by human every day. Bovine milk contains approximately 3.5% protein, of which 80% are caseins and 20% are whey proteins. Casein genes of cattle are located in the q31–33 region of chromosome 6 and they form a cluster of four closely linked genes located in the following order: ?s 1 -casein, ?-casein, ?s 2 -casein, and ?–casein. The entire cluster is 200 kb in length. The ?s 1 -casein, ?-casein, and ?s 2 -casein genes are linked most closely, whereas the ?–casein gene (CSN3 ) is at least 70 kb away from others. Kappa casein represents about 12% of total casein in bovine milk. This protein consists of 169 amino acid residuals with a calculated molecular weight 19000 Dalton. Two kappa casein variants denoted as A and B have been found in Holstein milk. Alleles A and B of the CSN3 gene differ from each other in two amino acid substitutions, 136 Thr (A)/Ile (B) and 148Asp (A)/Ala (B). The nucleotide substitutions in this positions leads to create or loss restriction enzyme recognition site and using this we can identify different alleles of this gene. Blood samples obtained from a random sampling of dairy Holstein cows maintained in five farms located in Isfahan province (n=408) to investigate polymorphism of kappa casein gene. Number of samples varied from 65 to 96 among herds. DNA was extracted using salting out procedure. The quality and quantity of DNA determined by electrophoresis and spectophotometry. The 633 bp fragment of this gene amplified using specific primers by PCR. This fragment consists of a part of intron III (4 bp), exon IV (516 bp) and intron IV (113 bp). PCR product was detected by electrophoresis on 1% TAE agarose gel. Amplified fragment digested using Hind III restriction enzyme for 10 to 14 hours at 37 ? c. The genotype frequency of AA, AB, and BB were 0.667, 0.229, and 0.033, respectively. Allele A of the ? -casein gene occurred at a higher frequency than allele B. The frequencies of alleles A and B alleles were 0.817 and 0.183 respectively. The A allele in Herd 1 was more frequent than other herds but the A allele in Herd 3 was lowest frequency. The BB genotype in herd 4 had highest frequency whereas this genotype in herd 1 and 2 wasn’t observed. All five herds were in Hardy-Weinberg equilibrium. The Shannon and Nei’s index showed herd number 1 has lowest and herd number 3 has highest gene diversity among the studied herds. The polymorphism information content (PIC) was 0.254, indicate that this marker has medium power to separate different genotypes. In this study the association between kappa casein genotypes and productive and performance traits was analyzed by MIXED procedure of SAS software. The results showed significant associations between the genotypes, with fat and protein percentages and lactation period. No significant association was found between genotypes and adjusted 305 day milk, pregnancy length, conception rate and SCS (P 0.05). Animals carrying the AB genotype had higher fat and protein percentages and longer lactation period than AA animals (P 0.05). It could be concluded that if the purpose of selection is increase of fat and protein percentage, we could deserve selection using this candidate gene. Key words: kappa casein gene, polymorphism, Holstein cow, Production Traits, genotype