K-casein gene was regarded as a candidate gene for milk production traits of cows. In this study, a 779 bp fragment of k-casein gene of Chinese Holstein was amplified by polymerase chain reaction (PCR), the polymorphisms of three loci of k-casein gene were detected by PCR-RFLP with restriction endonuclease Taq, Hind, Pst. After sequencing, T/C single nucleotide polymorphism (SNP) was identified at nucleotide 10 891C/A SNP was identified at nucleotide 10 927 and G/A SNP was identified at nucleotide 10 988 in exon4 of k-casein gene. Both alleles (A and B) of three loci were found in the population that showed low polymorphism. The gene frequencies of A and B were 86.03% and 13.97%, respectively. The genotype frequencies of AA, AB, and BB were 73.71%, 24.63%, and 1.66%, respectively. Statistical results of c2 test indicated that three polymorphism sites in the population fitted with Hardy-Weinberg equilibrium (P > 0.05). Meanwhile, the effect of polymorphism of k-casein gene on milk production traits was analyzed. The results indicated that in the three loci, the different genotype of k-casein gene had no significant influence on milk yield and milk protein percent (P > 0.05). The cows with genotypes BB and AB showed higher milk fat percent than those with genotype AA ( P < 0.05 ) ; with genotype AB showed higher fat protein ratio than those with genotype AA ( P < 0.05 ). The polymorphism of the three loci in the experimental population is closely linked. The conclusion is that k-casein B allele can be used as the molecular genetic markers of modifying milk fat percent in Chinese Holstein cows.