As a candidate gene related to egg production, prolactin plays a crucial role in the reproduction of birds. In this study, polymorphism of the prolactin gene was screened in six Chinese native duck breeds (Shanma, Shaoxing, Youma, Jinyun, Jingjiang and the F₂ resource population of white Liancheng X white Kaiya) using PCR-SSCP and direct sequencing. The results showed that 12 SNPs were detected: C-213T in the 5′flanking region, A-1842G in exon 2, A-3869G in exon 4 and C-5961T in exon 5, T-295C, C-381A and A-412G in intron 1, T-2231C in intron 2, C-3949T, T-3988G and T-4009C in intron 4 and T-6052A in the 3′ flanking region. The C-381A site and C-5961T site can be detected by XbaI and Pst1 PCR-RFLP, respectively. Furthermore, the C-5961T mutation results in an amino acid conversion (Cys to Arg) in the functional domain and affects the existence of a heparin binding site (L-R-R-D-S-H-K). In the F₂ resource population, association analysis demonstrated that the C-5961T polymorphism was significantly associated with egg production and egg weight, with the CC genotype associated with higher egg production and bigger egg weight than the CT genotype.