Kappa casein gene polymorphism has received a considerable attention because of its correlation with milk quality, composition and technological properties. The polymorphism of kappa casein gene (K-CN) was detected in Holstein Chinese cattle. A 218 bp sequence in exon IV of 319 Holstein Chinese cattle blood samples were amplified using polymerase chain reaction-single strand conformation (PCR-SSCP) technique. Sequence analysis revealed one single nucleotide polymorphism (SNP) T/C SNP in exon 1V at nucleotides (80), moreover; three genotypes TT, TC and CC were also identified with following frequencies: 0.40, 0.34 and 0.26%, respectively. The allele frequency for T and C found to be 0.6 and 0.4 %, respectively. Allele frequencies in the population fitted with Hardy–Weinberg equilibrium (P>0.05). Analysis of genetic polymorphism of k-casein at exon 1V exhibited medium polymorphism information content (PIC=0.36).