The population genetic structure of a reef-dwelling aeolid nudibranch, Pteraeolidia ianthina (Angas, 1864) (Gastropoda, Mollusca), was investigated by analyzing the nuclear ribosomal RNA gene region, including 18S and 5.8S rRNA genes and the ITS1 region (545 bp). Among 235 individuals from 10 localities in the northwestern Pacific, two genetically distinct groups were detected: Groups A and B, which are separated by a minimum sequence difference of 5.3% (p-distance). The marked genetic differentiation of these two groups, even in localities where both co-occur, suggests that they are distinct species, although intergroup hybrids were observed in very low frequency. Within-population genetic variability was low in Group A, but high in Group B. Geographic analysis of between-population genetic distance and “allele” sharing data indicated three subgroups within Group A (temperate Japan, Ryukyus Philippines, and Guam), whose distribution ranges appear to have some relationship to climate zones (temperate and subtropics tropics) and ocean currents (Kuroshio and North Equatorial Currents). Within Group B detected only from three subtropic and tropic localities, however, genetic distances between populations were much smaller than those in Group A, and thus no significant geographic pattern was found. These differences in genetic variability and population structure between Groups A and B may reflect differences in biological features between them, such as the duration of the pelagic larval stage. Furthermore, some morphological differences were observed between Groups A and B. These observations provide further support for the suggestion that the two groups are separate species.