Semisulcospira is a freshwater snail genus highly divergent in the ancient Lake Biwa, Japan, with a history of ~4 million years. Although the shell morphology, karyotype and molecular phylogeny of the genus have been well studied, the systematic status of several non-monophyletic species remains uncertain. In this study, we have evaluated the taxonomic accounts of the species previously identified as Semisulcospira decipiens, S. habei and relatives. We examined the genetic relationships using genome-wide SNP data and elucidated morphological variation among these using Random Forest classification. Morphological relationships between the name-bearing type of S. decipiens and the newly collected specimens were also evaluated. Morphological characteristics effectively discriminated between the nine genetic clusters, and the correlation among morphology and substrates was elucidated. We revised taxonomic accounts of S. decipiens, S. habei, S. arenicola, S. nakasekoae and S. ourensis and synonymised S. multigranosa, S. habei yamaguchi, S. dilatata under S. decipiens and S. fluvialis under S. nakasekoae. We also described two new species, Semisulcospira elongata sp. nov. and Semisulcospira cryptica sp. nov., and redefined two phylogroups of the lacustrine species as the Semisulcospira niponica-group and the Semisulcospira nakasekoae-group. Traits of the species examined exhibiting intraspecific variation in the different substrates and flow velocity may indicate the morphological and trophic adaptations. The habitat-related variation has certainly caused the taxonomic confusion of the lacustrine species. Lake drainage contributes to increasing the species diversity of the genus, generating ecological isolation between the riverine and lacustrine habitats.