AbstractElucidating the mechanism of formation of biota on islands will contribute to our understanding of the evolutionary mechanisms that generate biodiversity. In this study, we focus on the evolutionary history of Ephemera orientalis, which is distributed in East Asia. Phylogenetic analyses were performed using the mitochondrial DNA 16S rRNA, cytochrome oxidase subunit I and the nuclear DNA histone H3 regions. We found that these mayflies exhibit genetic differentiation between the Japanese and Korean populations. The Tsushima population, which is located between the Japanese Islands and the Korean Peninsula, was positioned genetically as a continental clade. The populations of the Fuji-Goko Lakes and Lake Ashino-ko in Central Japan were genetically differentiated from the other populations. The genetic structures over a wide area of the Japanese Islands were confirmed and a loose genetic regionality was observed, which was thought to result from secondary contact of the north-eastern and south-western lineages after their long separation by a geohistorical division event. We have gathered important knowledge regarding the evolutionary history of organisms on islands and investigated the colonization and isolation of biological populations within a species with high dispersal ability.