We compared the genetic diversity of the Lake Kasumigaura population of Nymphoides indica with that of pond populations in Hyogo and Kagawa Prefectures, which are thought to maintain high genetic diversity, to elucidate the current genetic diversity and occurrence of distinctive alleles in the Lake Kasumigaura population. The genetic diversity, as measured by the mean number of alleles per polymorphic locus, effective number of alleles per locus, mean observed heterozygosity, mean expected heterozygosities, total gene diversity, and number of multilocus genotypes was lower in the Lake Kasumigaura population than in the Hyogo and Kagawa populations. In addition, the inbreeding coefficient suggests that random mating does not occur in the Lake Kasumigaura population. The degree of genetic differentiation between the Lake Kasumigaura population and the Hyogo and Kagawa populations suggests that the Lake Kasumigaura population is largely genetically distinct. We found five genotypes in the Lake Kasumigaura population that were absent from the Hyogo and Kagawa populations. These results demonstrate that the Lake Kasumigaura population is an important component of the overall genetic diversity of N. indica in Japan.