Milk fatty acid composition is a parameter of great interest for evaluation of nutritional quality of milk. Stearoyl-CoA desaturase 1 ( SCD1 ; also known as ?9- desaturase) is a key enzyme in mammary lipid metabolism because it is able to add a double bond in the ?9- position in a large spectrum of medium and long chain fatty acids. With regard to the role of SCD1 in lipid metabolism and QTL studies for milk production traits, SCD1 has been regarded as a candidate gene related to milk production traits in dairy cattle. One polymorphism () in exon 5 region of this gene might control translation or stability of SCD1 mRNA. This polymorphism is the result of substitution of valin (allele V) with alanine (allele A). In the case of being alanine, there will be a restriction site for the restriction enzyme NcoI and as a result, alleles A and V will be recognizable as bands with length 200bp and 400bp respectively. To investigate this polymorphism and its relationship with milk production traits, blood sample from 408 Holstein cattle in 5 industrial dairy farms located in Isfahan province that had records for milk production and composition for first parity was randomly taken. Genotyping was done by PCR-RFLP technique. After extracting DNA, to determine the quality and quantity of extracted DNA, amplification and digestion by restriction enzyme, digestion product was observed on 1.5% agarose gel. Both alleles A and V were observed in all 5 herds and which their frequencies were 0.58 and 0.42 respectively. Frequency of genotypes AA, AV and VV were also 0.26, 0.65 and 0.09 respectively. The frequency of genotypes was not in Hardy–Weinberg equilibrium. Disequilibrium shows Herds could be under selection and the selection may indirectly affect SCD1 gene which cause disequilibrium for milk production and protein percentage. Genetic analysis of the population was done by the PopGen32 software. Results indicate a high diversity within this polymorphism and a high inbreeding and genetic relationship within the studied population. To investigate the effect of genotype on production traits, SAS software (procedure GLM) was employed. In analysis: the effects of genotype and combined effect of herd, year and season of parturition were considered as fixed effects and days in milk and open days were considered as co-variables. Genotype had significant effect on corrected milk production and protein percentage (P 0.01). Individuals with genotype VV had statistically more milk protein percentage and corrected milk production in comparison with genotypes AV and AA. Also cows having genotype AV showed more milk protein percentage and corrected milk production compared to genotype AA. These findings show that this polymorphism could be considered as an additive gene action. Dominance effect for the studied traits was not significant (P 0.05) which confirmed additively of the polymorphism. As there were a good relationship between the polymorphism with protein percentage and corrected milk production and also a high diversity within the studied population, this polymorphism can be used as a selection tools for these traits.