A simple reliable method using multiplex PCR with species-specific primer sets was developed for identification of three Pacific Hediste species ( Polychaeta: Nereididae), which are morphologically indistinguishable in sexually immature specimens. They commonly inhabit estuaries in eastern Asia (H. atoka and H. diadroma) or the western coast of North America (H. limnicola). The species-specific oligonucleotide primers were designed based on interspecific polymorphism of mitochondrial 16S rRNA genes. The three Hediste species were distinguishable according to their electrophoretic patterns showing different molecular sizes of the species-specific PCR Products. This method is superior, both in speed and lower cost, to the PCR-RFLP method we proposed previously.