After analyzing the coding sequence (CDS) of chicken prolactin gene by DNA sequencing, one point mutation of C1607T in exon2 and two mutations of C5749T and T5821C in exon5 were discovered. Although none of the mutations changes amino acid encoded, they have different codon frequencies in the five populations. The three mutations in 370 individuals of the five populations by single-strand conformation polymorphism (SSCP) were screened, and seven haplotypes in the CDS of prolactin were discovered. Moreover, the seven haplotypes show different high frequency value, which was defined by the number of high frequency codon of the haplotype. Analyzing the laying performance of the five breeds, the positive correlation between the laying performance and high frequencies value wea found. After detecting the expression of the prolactin by enzyme linked immunosorbent assay (ELISA), it was found that the higher number of high frequency condon will lead to the more quantity of prolactin. The results demonstrate that the codon frequency is a factor which influence the laying performance, and synonymous mutation might influence phenotype by changing codon frequency.