The polymorphisms of 5' flanking region of chicken prolactin (cPRL) gene were examined in several populations of Chinese native Yuehuang, Taihe Silkie and imported White Leghorn Layer chickens. The 5' flanking regions (2638 bp) from Yuehuang, Taihe Silkie and White Leghorn chickens were subjected to sequencing analysis. Four single nucleotide polymorphisms (SNPs) were identified at position -2425(C/T), -2215(T/C), -2063(G/A) and -1967(A/G). A 24-bp indel (insertion or deletion) and a polyA length polymorphism were also identified. For the 24-bp indel locus, three genotypes (AA, AB and BB) were found in Yuehuang chickens, while only two genotypes were detected in Taihe Silkie (AB and BB) and Leghorn chickens (AA and BB). The genotype frequencies of AA, AB and BB were significantly different among the three breeds. For the polyA locus, although three genotypes (CC, CD and DD) were found, only one genotype (CC) was detected in White Leghorn chickens, while two or three genotypes were observed in Chinese native chickens. We used real-time quantitative PCR and radioimmunology assay to investigate the potential association of the 24-bp indel locus with cPRL mRNA expression, plasma cPRL and brooding behaviors, and observed that chickens with genotype AB, which are of the highest incidence of broodiness, had the highest cPRL mRNA levels, providing the possibility that this polymorphic site might be related to the broodiness in chickens via modulating the transcriptional level of cPRL gene. The dissociation among cPRL gene transcription, mRNA storage and hormone release was also observed.