ABCG2 protein or Breast Cancer Resistant Protein (Bcrp), located in membrane of mammary glands alveolar epithelial cells, is one of the most important active traort proteins and responsible for passing of fat from blood to milk. It is encoded by ABCG2 (Bcrp). These traort fat are very important for veterinary medicine. Single nucleotide polymorphism, which is caused by changing amino acid (Y581S), in ABCG2 gene that encodes Bcrp, affect milk yield and milk properties in Israeli Holstein. In the case of being nucleotide C, there will be restriction site for the restriction enzyme Pst I and as a result, alleles A and C will be recognizable as bands with length 292bp and 268bp respectively. To hnvestigate this polymorphism and its relationship with milk production traits, blood sample was randomly taken from 408 Holstein cattle in 5 industrial dairy farms located in Isfahan province that had records for milk production and its composition for first parity. Genotyping was done by PCR-RFLP technique. After extracting DNA, determining the quality and quantity of extracted DNA, amplification and digestion by restriction enzyme, digestion product was observed on 2.5% agarose gel. Frequencies of all A and C werw 0/97 and 0/03 respectively totally. Frequency of genotypes AA and AC werw also 0/94 and 0/06 respectively. On the other hand CC homozygous had not been found in investigated animals. All of them were in Hardy–Weinberg equilibrium. Being in equilibrium can be owing to selecting animals according not to ABCG2 gene. Genetic analysis of the population was done by the software PopGen32. Resulted indices indicates high diversity of this polymorphism and high inbreeding and genetic relationship in the studied population. To identify the effect of genotype on production traits, the software SAS, procedure GLM was used. In the analysis model the effects genotype and combined effect of herd, year and season of parturition were considered as fixed effects and days in lactation and open days were considered as covariables. Genotype had significant effect on fat and the breeding value of milk production (P 0/001). Individuals with genotype AC had statistically more milk fat percentage and genotype AA breeding value of milk production. This finding can be a reason that the effect of this polymorphism is additive. For trait milk fat percentage, the average effect of substitution of allele C was significant (P 0/001), but dominance effect for the studied traits was not significant (P 0/05) which indicates the effect of the polymorphism as additive. Regarding the relationship between the polymorphism and traits milk fat percentage and the breeding value of milk production in the studied population, the polymorphism can be used is selection for these traits. Key words: ABCG2 Gene, Holstein Cattle, Genotype, Polymorphism