Prolactin (PRL) plays central roles in mammals' reproduction, gland development, milk secretion, and the expression of milk protein genes. In dairy cattle, the PRL gene is a potential quantitative trait locus and genetic marker related to milk performance traits. Here, a total of 586 randomly selected Chinese Holstein cows were genotyped for locus PRL-RsaI. One haplotype block containing eight SNPs was identified in the region from intron 3 to intron 4 of the PRL gene in Chinese Holstein cows. One tag SNP (7545 G → A) was selected to represent the haplotype block defined by the genotypic data. The cows with genotype AA of this tag SNP had a higher milk yield at 305 days (8457 ± 938 kg) than the cows with GA (7537 ± 1278 kg; P < 0.01) or GG (7757 ± 1174 kg; P < 0.05). This suggests that the haplotype block examined in this study contains important markers for milk production traits in Chinese Holstein cows.