The tenthredinid sawflies Macrophya apicalis and M. infumata, which range throughout Japan and adjacent areas, are very similar in external morphology and share the same host plant, Sambucus sieboldiana. We investigated their genetic distances within the genus and their intraspecific variation, and estimated their time of divergence to clarify the history of their speciation and colonization. We collected 33 specimens of the two species from several locations in the Japanese archipelago (Hokkaido, Honshu, Shikoku, and Kyushu) and Cheju Island (Korea). We extracted total DNA from each specimen, and PCR amplified and sequenced 1300 bp of the cytochrome oxidase subunit I (COI) gene. We constructed phylogenetic trees by using the UPGMA and maximum likelihood methods. We estimated divergence times based on previously published COI substitution rates. The genetic distance between the two species indicated that their ancestors differentiated in the late Miocene, probably on the Asian continent. Based on the calculated time of intraspecific diversification in Japan, we estimated that both species immigrated to Japan in the early Pleistocene and crossed the Tsugaru Strait land bridge between Hokkaido and Honshu after the middle Pleistocene. Genetic analysis of the two sibling species elucidated their relative taxonomical positions within the genus, and their intraspecific variation. Estimated from previously published COI evolutionary rates, the time of arrival of both species in Japan is consistent with the history of colonization of the country by other animals during the last ice age.