The objective of this study was to determine the genetic polymorphism of prolactin in Montebeliard cattle, unique kind in Iran in Behkedeh-e-Razavi located at northern Khorasan breed and its association with milk production. Blood samples were taken from 120 samples and DNA was extracted. A fragment of prolactin gene with the length of 156 bp including exon and intron3 was amplified by PCR and RFLP using RsaI enzyme. Frequency of allele A and B Was 0.89 and 0.11 respectively. The chi- square test was used to check for Hardy-Weinberg equilibrium at this locus. It was found that the population was in equilibrium at these restriction sites. Association between the marker genotypes and milk production trait was analyzed by SAS and MATLAB software. It was observed that The AA genotype group yielded most milk in compared of other groups. There was no significant difference between milk yield of AB and BB genotype groups. This genetic polymorphism could be used for biotechnology and modified and correct milk product.