In the mid-1990s, Neoheterobothrium hirame suddenly appeared as a new species in olive flounder Paralichthys olivaceus in Japanese coastal waters. Anemia caused by the parasite has prevailed in wild and cultured populations of olive flounder since that time. In this study, to clarify the origin of N. hirame, two Neoheterobothrium species, namely unidentified Neoheterobothrium species (tentatively abbreviated as Neoheterobothrium sp. PL) and N. affine, were collected from Paralichthys lethostigma and Paralichthys dentatus, respectively, off the east coast of North America and compared with N. hirame collected in Japan. No substantial differences were detected in the morphology and DNA sequences of ITS1-5.8S rRNA-ITS2 and mitochondrial cytochrome oxidase subunit I (mt COI) regions between N. hirame and Neoheterobothrium sp. PL. On the other hand, the congeneric N. affine was clearly distinguished from both N. hirame and Neoheterobothrium sp. PL in its longer isthmus and the DNA sequences in ITS1 and mt COI. The absence of differences between N.hirame and Neoheterobothrium sp. PL and the clear difference between both of these and N. affine indicate that N. hirame is conspecific with Neoheterobothrium sp. PL infecting P. lethostigma and that N. hirame was recently introduced from North America to Japan.