Lake Biwa (Shiga Prefecture, Japan) is an ancient lake with many endemic species. In particular, 15 extant endemic species of the genus Semisulcospira (Caenogastropoda: Pleuroceridae) have attracted attention because they may represent a single species flock. Although many taxonomic studies have been conducted on these Lake Biwa species and on non-Biwa species of the genus, their genetic relationships remain unresolved. We use allozyme analysis to investigate the genetic differentiation of 12 species of the genus Semisulcospira, including nine Lake Biwa endemics: S. (Biwamelania) arenicola Watanabe and Nishino, 1995, S. (B.) decipiens (Westerlund, 1883), S. (B.) fluvialis Watanabe and Nishino, 1995, S. (B.) multigranosa (Böttger, 1886), S. (B.) nakasekoae Kuroda, 1929, S. (B.) ourense Watanabe and Nishino, 1995, S. (B.) fuscata Watanabe and Nishino, 1995, S. (B.) niponica (Smith, 1876), and S. (B.) habei Davis, 1969, and three non-Biwa species: S. (S.) libertina (Gould, 1859), S. (S.) reiniana (Brot, 1877), and S. (S.) kurodai Kajiyama and Habe, 1961. Based on their genetic features, the Lake Biwa endemics were divided into three groups, the S. (B.) decipiens group, S. (B.) niponica group, and S. (B.) habei group. The observed genotype frequencies of the Lake Biwa endemics within each group were similar to each other. We tested the observed genotype frequencies for each polymorphic locus in each local population of the Lake Biwa endemics for departure from Hardy—Weinberg equilibrium. Most of the eight polymorphic loci of the Lake Biwa populations did not deviate significantly from HWE, except for Aat of S. (B.) arenicola at the site B8, Pgm-1 of S. (B.) fluvialis at the site B9, and S. (B.) habei at the site B1 (level of significance α = 0.05). The S. (B.) niponica and S. (B.) habei groups showed similar genetic features to the non-Biwa S. (S.) libertina and S. (S.) reiniana groups, whereas the S. (B.) decipiens group was distinct from any other group. Our allozyme data suggest that Semisulcospira species in Japan are distinguished robustly into two major lineages, the S. (B.) decipiens group and a separate non-decipiens group, and that all extant Lake Biwa endemics do not form a single species flock, as proposed previously.