In East Asia, the mayfly genus Ephoron, a member of Polymitarcyidae, has been classified into three species: Ephoron eophilum, Ephoron limnobium, and Ephoron shigae. Using the nuclear histone 3 and internal transcribed spacer 1 and mitochondrial 16S rRNA and cytochrome c oxidase subunit I genes, we investigated the phylogenetic relationships of these mayflies. Ephoron limnobium formed polyphyletic relationships within the clade of Japanese E. shigae and shared identical sequences with E. shigae, suggesting that E. limnobium is a synonym of E. shigae. Monophyly was supported in E. eophilum, Korean E. shigae, and E. limnobium + Japanese E. shigae. Ephoron eophilum that co-occurs with the other Ephoron species has several peculiar traits in its adult swarming period and karyotype. This suggests that both pre- and post-zygotic isolation are likely to have promoted speciation of these Ephoron species. The divergence between E. eophilum and E. shigae would have occurred from the late Late Miocene to Early Pliocene according to the Bayesian relaxed clock model calibrated using the mitochondrial substitution rate. The area currently inhabited by E. eophilum emerged above sea level some 0.1 Mya, suggesting that E. eophilum moved into this area from other areas that it occupied prior to this time. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 181–202.