The pituitary-specific transcription factor (PIT1) gene is responsible for pituitary development and hormone secreting gene expression in mammals. PIT-1 is studied as a candidate genetic marker for growth, carcass and also for milk yield traits. In dairy farm animals, the principal goal of the selection is the improvement of milk yield and composition. The genes of milk proteins and hormones are excellent candidate genes for linkage analysis with quantitative trait loci (QTL) because of their biological significance on the quantita-tive traits of interest. Thus, in this study association between polymorphism of the pituitary transcription factor 1 (PIT1) gene and milk fat percentage of HOLSTEIN CATTLE in Khorasan Razavi province of Iran were analyzed. A total of 100 dairy cows from a herd containing 1000 animals were included in the study. Genomic DNA was extracted from the whole blood. One pair primers were used for amplification of PIT1 gene and PCR products were electrophoresed on 1% agarose gel. Then, PCR products were digested with HinfI restriction enzyme. Results were analyzed using PopGene software and allele frequencies A and B were 0.25 and 0.75, respectively. Frequencies of AA, AB and BB genotypes, number of observed alleles, number of effective alleles, expected heterozygosity, observed heterozygosity, mean of heterozygosity, expected hemozygosity, observed hemozygosity, Nei’s index and Shanon’s index were 6, 40 and 54%, 2, 1.6, 0.37, 0.40, 0.37, 0.62, 0.59, 0.37 and 0.56, respectively. Results of k-square shown that population is in Hardy-Weinberg equilibrium. SAS software with GLM procedure was used for calculation of association between milk fat percentage and observed genotypes and results indicated that the effect of genotype on fat percentage was significant (P<0.01) and AB genotype had the highest effect on milk fat percentage. These results imply that the PIT1 genotypes affected milk fat percentage, suggesting that this polymorphism can be used as a molecular marker for this trait.