Abstract: Toll-like receptor 2 (TLR2), a key component of the innate immune system, plays an important role in the initiation of the inflammatory response to foreign pathogens. This study was carried out to investigate the TLR2 gene expression difference in mammary gland tissues of the cows infected with mastitis and healthy cows and to search for variants of the bovine TLR2 gene for further clarifying the relationship between TLR2 gene and mastitis in cattle. The quantitative real-time polymerase chain reaction (qPCR) revealed that clinical mastitis caused by Staphylococcus aureus could significantly increased the LTR2 mRNA abundance (p<0.05). Seven novel single nucleotide polymorphisms (SNPs) in the 5’ upstream of the TLR2 gene in six cattle breeds (Hereford, Simmental, Limousin, Chinese indigenous Luxi Yellow cattle, Bohai Black cattle and Chinese Holstein cattle) were identified by the direct sequencing method. Four SNPs are located at the putative promoter region and their variants can alter the transcriptional factor binding sites. Genetic diversity showed that the SNP (4bp deletion) in the promoter was unique and two distinct haplotypes were found in Hereford cattle. Six SNPs in the TLR2 gene exon2 and 3’UTR were genotyped by PCR-RFLP and CRS-PCR methods in 398 Chinese Holstein cattle. Case-control study and associated analysis revealed that the cow with the genotype TT (3.15±0.52) in c.+189T>G has significantly lower (p<0.05) somatic cell score (SCS) than that with genotype TG (4.84±0.21) or GG (5.31±0.27) in Chinese Holstein cattle. In conclusion, haplotype analysis and linkage disequilibrium findings showed that the hapoltype TG of two loci (c.+189T>G and c.+631A>G) in the coding region may be used as a tolerance haplotype for the bovine mastitis.