Prolactin (PRL) have been showed a potential candidate gene for growth and reproductive traits in chicken and ducks that have revealed in many works. Thus, the aims of this study were to evaluate the polymorphism of prolactin gene in crossbred duck (Star53xBien). A totally 60 blood samples were collected for conducting PCR-RFLP. Genomic DNA was isolated by ABT kit (Vietnam). Based on the purified genomic DNA, PCR-RFLP was applied. A total of 60 crossbred ducks were genotype for HphI và Xbal restriction site. The result indicated that the monomorphic was found at PRL/Hphl site. The polymorphic site at PRL/Xbal was observed and allele frequencies for T and G were 0.89 and 0.11. Two genotypes were observed and the genotype frequencies were 0.78 and 0.22 for TT and TG/Xbal, respectively. Additionally, the PIC and expected heterozygoty were 0.177 and 0.196, respectively. In conclusion, the polymorphic site at locus PRL/Xbal was detected and T allele and TT genotype were dominant. In order to understand more insight the association of PRL/Xbal with growth and reproductive traits of crossbed duck, further study is required.