In Thailand, there are at least ten species of freshwater snails of the family Bithyniidae, most of which can act as intermediate hosts of veterinary and medically important parasites. The genetic variation, geographical distribution, and taxonomic status of some species are obscure, including for Hydrobioides nassa. Thus, this study aims to explore the genetic variation and distribution of H. nassa in north, west, and central Thailand. We collected 264 specimens of H. nassa from 46 localities in 16 provinces using mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal DNA sequences to determine variation. Genetic diversity of H. nassa is relatively high, with 65 and 11 haplotypes of COI and 16S rDNA observed, respectively. The phylogenetic tree and haplotype network analyses classified H. nassa into three haplogroups (haplogroup I–III). Haplogroup I and II belonged to clade A, whereas haplogroup III belonged to clade B. Interestingly, haplogroup III or clade B contained the specimens from Yom river basin in Phrae province, which was the most genetically distinct. Thus, this study suggests that H. nassa in Thailand is a complex of phenotypically similar but genetically distinct species.