Toll-like receptor 4 recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 477 bp segment of the 5′-flanking region of TLR4 gene of Chinese Holstein, Sanhe cattle and Chinese simmental was amplified by polymerase chain reaction. After sequencing, a polymorphic site in amplified production of TLR4 was identified of having either a G or a C at position 245. This polymorphism in the three populations was detected by digesting the fragment with restriction endonuclease MspⅠ. Results showed that both alleles(A and B) were found in the three populations and the value of polymorphism information content indicated that this was a moderate polymorphism. c2 test indicated that the polymorphism locus in Sanhe cattle did not fit Hardy-Weinberg equilibrium (P < 0.05). In addition, the effect of the TLR4 polymorphism on somatic cell score was analyzed, and the results indicated that the somatic cell score were significantly affected by lactation month and the type of breeds(P<0.05), but not by different genotypes (P > 0.05).