AbstractThe goal of this study was to explore the level of Single Nucleotide Polymorphisms (SNP) level of 5′-flanking regions in chicken functional genes. The 5′-flanking region of eight genes, including GH, DDBC1, RIKEN, RASGRP3, IGF1, THRSP, VIP and PRL, were amplified and sequenced for White Leghorn, White Recessive Rock, Xinghua, Silkie, Gushi, and Red Jungle Fowl strains. A total length of 8,399 bp se-quence was obtained and 161 SNPs were identified, with an average of one SNP per 52 bp. Watterson’s esti-mate of θ was 0.00620 ± 0.00110 and the average nucleotide diversity (π) calculated from the average num-ber of pair-wise differences was 0.00559 ± 0.00100. The SNP diversity of 5′-flanking regions was signifi-cantly lower than that of introns. Because of their importance in regulation of gene expression, the 5′-flanking regions may have endured more selective pressure than introns during molecular evolution. A relatively low SNP diversity in 5′-flanking region may be more adoptive. The Tajima test and Fu & Li test showed that VIP and PRL, which are significantly associated with chicken productivity, are likely to be target genes for artificial and natural selection.