Abstract: The objective of this study was to study the genetic polymorphisms of two sites of κ-casein CSN3 gene in Jiulong yak. Genomic DNA was quickly extracted from the muscle of Jiulong yak bulls（n=100） by chelex 100 method and a partial sequence of exon 4 of κ-casein CSN3 gene was amplified by PCR. The polymorphic sites and their associations in the amplified sequence were analyzed by non-denaturing PAGE and PCR-SSCP methods. The results showed that the CSN3 gene of Jiulong yak bulls had a 12 bp repeat sequence polymorphism, which encoding 4 amino acids, resulting in 3 genotypes namely LL, LS and SS, and the genotype frequencies were 0.640, 0.280, 0.080, respectively, and the sequencing revealed the L allele had 12 bp duplication more than S allele. The established PCR-SSCP method could identify the C/T missense mutation at 13 064 bp of CSN3 gene, which showed different genotypes, namely CC, CT and TT, and their frequencies were 0.680, 0.250, 0.070, respectively. The L allele with 12 bp repeat sequence and the C allele encoding Thr₁₃₆ in CSN3 gene were significantly superior in Jiulong yak bulls. It indicated that the non-denaturing PAGE and PCR-SSCP method could accurately analyze CSN3 genotypes in Jiulong yak bulls, and the two polymorphic sites might be evolutionarily related.