Because of the TLR4 polymorphism on the milk trait has been proven, this study was to assess the frequency of TLR4 T4CRBR2 polymorphism in Iranian Holestein dairy cows. In addition, the other aim in this study was estimating the association between the TLR4 T4CRBR2 and productive trait performance of milk as well as somatic cell score of milk (SCS) in this population. The gene toll like receptor 4 (TLR4) had been mapped to the bovin BTA8 and encodes the TLR4 receptor. The considered polymorphism results from C à T nucleotide substitution in TLR4 gene exone 3 and so, teronine to isolusine of amino acid change in product protein. This polymorphism proved to significantly affect the percentage milk productin.TLR4 gene was studied in this population (400 cows) by PCR-RFLP technique. The 382 bp fragment including the considered polymorphism was amplified and digested with enzyme ALU1 to determine the genotypes of 400 Holstein cows. Both alleles A and B were detected after digested by the restrictive enzyme. AA individuals have not mutant alleles, AB individuals have one mutant allele and BB individuals have two mutant alleles. Genetic analysis of the population was done by the software PopGen32. Frequency of genetic AA, AB and BB genotypes were respectively 0.1, 0.55 and 0.35 and frequency of A and B alleles were estimated 0.37 and 0.63, respectively. For the statistical analysis investigation relationship between TLR4 T4CRBR2 with productive traits of milk and somatic cell score of milk in first lactations, the software SAS,procedure GLM was used. In the analysis model the effects of genotype and combined effect of herd, year and season of parturition were considered as fixed effects and days in lactitation, open days and milk production were considered as covariables. The statistical analysis showed significant effects of the genotype for corrected fat percentage, milk breeding value, protein percentage and somatic cell score of milk traits but genotypes have not significant effect for fat breeding value. Estimated average effects of substituting the isoleusine for the tereonine variant on record corrected of milk production, dairly milk production average, fat percentage, protein percentage, somatic cell score of milk and on milk breeding value and fat breeding value. These results can be showed that allele B is effect on increase milk production and decrease fat of milk. The relatively hight frequency of the B allele than the A allele may be due to selection for milk yield in recent years. Results showed that the TLR4 T4CRBR2 polymorphism can be a source that underlies the reported quantitative trait loci for fat content trait in the proximal region of bovine chromosome 8. Keywords: TLR4 gene, Holstein cows, Genotype, Polymorphism