By the technology of polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),genetic polymorphism of exon 4 and exon 5 of κ-casein gene in southern Chinese Holstein cattle were studied.The results showed as follows: exon 4 and exon 5 of κ-CN gene were polymorphic caused by a AC substitution in exon 4(g.13100 AC) and a TC substitution in exon 5(g.5153 AC).The allele frequencies of A,B of exon 4(AA,AB) and exon5(AA,AB,BB)of κ-CN gene were 0.9379,0.0621 and 0.6512,0.3488,repetively.The genotype distribution of the two exons both fitted Hardy-Weinberg Law(P0.05). Exon 4 locus showed low polymorphism(PIC=0.1097) while Exon 5 locus showed normal polymorphism(PIC=0.3511).The heterozygosity and number of effective alleles were 0.1165,1.1318 and 0.4543,1.8324,respectively in exon 4 and exon 5 of κ-CN gene of southern Chinese Holstein cattle.