Quantitative trait loci (QTL) affecting milk production traits on bovine chromosome 6 close to the OPN gene location have been identified through several whole genome scans. OPN is a phosphoprotein, synthesized in a variety of tissues and cells and secreted into body fluids. In this study, DNA was isolated from blood samples collected from 398 Holstein cows in Tehran and Esfahan provinces. DNA isolation was performed using salting out method. RFLP-PCR method was used for genotyping of SNP C>T at position 8514 in OPN gene. Frequencies of CC, CT and TT genotypes were estimated as 19, 57 and 24%, respectively. Hardy-Weinberg Equilibrium (HWE) was evaluated using ? square test. The genotypes in this locus deviated from HWE. In this study, significant associations were observed between c.8514C>T genotypes in OPN gene and FATP2X, PROPER305 traits. The SNP, showing signi?cant association with milk traits would provide an excellent opportunity for marker assisted selection programs in dairy cattle.