Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using the newly developed environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. Among the B. toni eDNA-positive sample set, 51 samples were chosen to implement the Barbatula-specific eDNA assay on a fine scale with the goal of determining the phylogeographic pattern. As a result, two regionally restricted, genetically distinct lineages of the species were revealed. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonization of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.