The intermediate stages of speciation are important for understanding the processes involved in the creation of biodiversity, and also comprise a number of interesting phenomena. However, difficulties are associated with dividing clear speciation stages because speciation is a continuous process. Therefore, the elucidation of speciation is an interesting and important task in evolutionary biology. We herein present an example of a species in an intermediate stage of speciation using the giant water bug Appasus japonicus (Heteroptera, Belostomatidae) that was investigated using mating experiments and phylogenetic analyses of the mtDNA COI (658 bp) and 16S rRNA (435 bp) regions, and nDNA SSR (13 loci) and its genome-wide SNPs (11,241 SNPs). The results of our phylogenetic analyses based on their mtDNA dataset and the genome-wide SNPs dataset strongly supported the paraphyly of the Japanese populations. Therefore, it is suggested that their ancestral lineage which being distributed in the Japanese Archipelago subsequently migrated to the Eurasian Continent (i.e., “back-dispersal” occurred). Furthermore, the results of the mating experiments suggested that among A. japonicus, even between closely related lineages, pre-mating reproductive isolation has been established by the differentiation of copulatory organ morphologies. In contrast, pre-mating reproductive isolation is not established in the absence of the differentiation of copulatory organ morphologies, even if genetic differentiation is prominent. These results suggested that their phylogenetic distance does not predict pre-mating reproductive isolation. Furthermore, in the present study, we present a clear example of pre-mating reproductive isolation driving speciation between closely related lineages.