The aim of the present study was to investigate the effect of a single nucleotide polymorphism (SNP) of the endothelin receptor B subtype 2 (EDNRB2) gene on plumage coloration in mule ducks. Test mating (white Tsaiya × white Muscovy ducks) in combination with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to investigate the effect of non-synonymous SNPs in two maternal lines (a conservation and a selection population) on plumage coloration in mule ducks. One non-synonymous SNP (c.995G>A) was identified in white Muscovy ducks and white Tsaiya ducks by PCR-high-resolution melting (PCR-HRM) and DNA sequencing. Genotyping results showed that the c.995G>A locus is associated with plumage colour in two maternal populations of white Tsaiya ducks. Further, the maternal genotype of c.995G>A SNP affects the plumage colour of mule ducks. Therefore, the polymorphisms within the EDNRB2 gene at c.995G>A in white Tsaiya ducks may be used in marker-assisted selection to improve the plumage colour of mule ducks.