Four breeds of chickens (White Leghorn, Yangshan, Taihe Silkies, White Recessive Rocks) with different egg production were applied to screen potential single nucleotide polymorphisms (SNPs) related to reproduction trait in distal part region of prolactin gene based on DNA pooling and sequencing. Eight SNPs (C2402T, T-2192C, C-2161G, C-2134G, C-2062G, G-2040A, A-1944G and C-1884A) were found successfully in the fragment of 1 028 bp at the distal part of 5'flanking region. Moreover,a simple and rapid method to estimate allelic frequencies of SNPs in each breed by calculating the ratio of allele peak heights was introduced here. The accuracies of estimation for the allelic frequencies of C-2402T, C-2161G, C-1884A, C-2062G and G-2040A were verified by comparing with the results of PCR-RFLP or PCR-SSCP.