Aedes (Stegomyia) flavopictus Yamada is widely distributed in Japan and Korea. The species comprises three subspecies based on current morphological taxonomy: Aedes f. flavopictus in the Palearctic region of Japan, Ae. f. downsi Bohart & Ingram from Amami and Okinawa Islands, and Ae. f. miyarai Tanaka, Mizusawa & Ingram from Ishigaki and Iriomote Islands of the Ryukyu Archipelago. These subspecies designations are based on observations of a combination of several morphological characters, none of which, by itself is diagnostic for discriminating among the three subspecies. To further study the relationships in this group, we examined the nucleic acid sequence divergence in the internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA gene array of Ae. flavopictus individuals collected at five sites from three geographic regions in Japan. Analysis of sequence data by distance and maximum parsimony methods produced phylogenetic trees that showed separation of the specimens into three major clades, corresponding to both subspecies and geographic region. These results were consistent with and support the current classification and geographic distribution of the three subspecies.