A single nucleotide polymorphism of 5' flanking region of the prolactin gene was investigated in both high prolificacy breeds (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset and Suffolk sheep) using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). The results indicated that two genotypes (AA and AB) were detected in Small Tail Han sheep (n = 239), only one genotype (AA) was detected in Hu (n = 40), Dorset (n = 50) and Suffolk sheep (n = 39). The mutant homozygous genotype (BB) was not detected in four sheep breeds. In Small Tail Han sheep (n = 239), the frequency of genotypes AA and AB was 0.91 and 0.09, the frequency of the A and B alleles was 0.95 and 0.05, respectively. The fitness tests showed that the Small Tail Han sheep population was in Hardy-Weinberg equilibrium. Sequencing revealed a mutation (G-->T) at the position 63 bp of the 5' flanking region of prolactin gene in AB genotype compared with AA genotype in Small Tail Han sheep. The Small Tail Han ewes with AB genotype had 0.83 (p < 0.05) lambs more than those with AA genotype. These results preliminarily showed that the prolactin locus is either a major gene that influences the high prolificacy in Small Tail Han sheep or is in close linkage with such a gene.