The East Asian giant water bug species Appasus japonicus Vuillefroy and Appasus major Esaki are aquatic hemipteran insects whose ranges overlap, particularly in the Japanese Archipelago and on the Korean Peninsula. In rare cases, the two species co-occur. Furthermore, they are very similar ecologically and also morphologically, making their identification extremely difficult, and the possibility of hybridization has also been suggested. In the present study, we re-examined their taxonomic validity, and the characteristics useful for identifying them. To re-examine the morphological traits useful for distinguishing these two species, 222 specimens of A. japonicus collected from Japan, Korea, and China, and 132 specimens of A. major from Japan and Korea, were examined. We also performed molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I (COI) regions and the nuclear DNA Histone 3 region. Although the two species are very similar ecologically and also morphologically, they showed significant genetic differentiation. Thus, there is likely some form of reproductive isolation acting between them. Major morphological characteristics overlap extensively between A. japonicus and A. major, and no particular trait was identified as being effective for differentiating these species. All the morphological characteristics examined overlapped between A. japonicus and A. major. However, a principal component analysis based on all of the morphological characteristics revealed that, despite the overlap between these species, it was possible to morphologically distinguish them. Therefore, a more accurate identification becomes possible using multiple characteristics rather than a single characteristic. The male genital paralobes, evaluated as the most useful morphological characteristic, was effective with 100% probability for the Japanese Appasus species. However, for the Asian (i.e. Korean) specimens, this characteristic was not useful. On the other hand, the results of molecular phylogenetic analyses based on the mitochondrial DNA 16S rRNA and COI regions and the nuclear DNA Histone 3 region clearly showed significant genetic differentiation between the two species. Notably, the results for the mitochondrial COI region strongly supported the independence of each monophyletic group (i.e. validity of each species). Therefore, DNA barcoding based on the mitochondrial DNA COI region is also considered useful for the identification of A. japonicus and A. major. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110, 615–643.