Semisulcospira freshwater snails are a prominent element of the freshwater macrozoobenthos throughout East Asia and have undergone substantial diversification throughout Japan, particularly in ancient Lake Biwa. Up to 19 species are currently recognized in Japan, including a large number of endemics in Lake Biwa that are placed in the subgenus Biwamelania. However, previous workers have encountered pervasive polymorphism in morphological characters, rendering current species hypotheses tentative. Based on extensive sampling of populations from throughout Japan, followed by genetic characterization with one nuclear (28S rDNA) and two mitochondrial gene fragments (cytochrome c oxidase subunit I and 16S rDNA), the phylogenetic lineage differentiation of Semisulcospira in Japan is studied for the first time in an attempt to delineate species boundaries. The phylogenetic analyses revealed insufficient differentiation in 28S to address phylogenetic relationships amongst the Japanese species. While the mtDNA markers revealed substantial lineage differentiation, there was wide-ranging incongruence between the topology of the mtDNA-based trees and morphospecies. Moreover, there was no discernible phylogeographic structure. Haplotypes from the main mtDNA clades were found to be represented in nearly all Japanese regions, with Lake Biwa being a reservoir for every mtDNA clade. I conclude that mtDNA markers are unsuitable to address species limits in Semisulcospira from Japan and discuss polymorphisms in morphology and mtDNA plus introgression as possible causes for the observed incongruence, but without being able to identify its ultimate source.