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The prolactin (PRL) gene has previously been reported as a potential candidate for egg production in birds. This study identified the prolactin gene polymorphism in the exon 5 region and its association with reproductive traits of TB crossbred ducks. Three hundred and thirty-one crossbred ducks were used in this study, with blood samples collected from the wing vein for DNA extraction to amplify the fragment length with 536 bp by PCR, and then the PCR products were cleaved with the PstI enzyme. The fragment length with 536 bp was successfully amplified in all individual samples, while genetic polymorphism at the PRL/PstI site was identified as C and T alleles, with allele frequencies 0.811 (C) and 0.189 (T). Three genotypes were observed with frequencies 0.647, 0.329 and 0.024 for CC, CT and TT, respectively. The PIC (polymorphic information content) and He (expected heterozygosity) were 0.260 and 0.306. The group of ducks with CC/PstI genotype laid the first egg significant earlier (P< 0.05) than CT/PstI or TT/PstI genotype ducks (145.3 vs 152.3 days or 153.2 days, respectively), the total of egg number up to 38 weeks of age was higher (P< 0.05) in the group of ducks with CC/PstI genotype compared to CT/PstI or TT/PstI genotype (99.3 vs 93.1 or 91.7 eggs). The average egg weight did not significant difference among genotypes (72.3, 72.2 and 70.6 g; P> 0.05). From these results, the polymorphic site at PRL/PstI was identified, ducks bearing CC genotype possess earlier age at first egg and egg yield up to 38 wks of age and considered as a candidate gene for supporting genetic selection in ducks to improve egg production.