Since primary freshwater fishes depend exclusively on each freshwater system throughout the life histories, their phylogeographic structures often reflect historical processes of diversification and distribution. However, traditional phylogeographic approaches require a great deal of effort to collect many DNA samples from large geographical areas. In this study, we conducted a large-scale environmental DNA (eDNA) survey in Hokkaido to understand the geographic patterns of both species distributions and intra-genetic variations of freshwater fishes. Firstly, the species distributions were comprehensively evaluated by MiFish eDNA metabarcoding. Based on the metabarcoding result, we focused on the most-commonly detected fish, Siberian stone loach (Barbatula toni) and applied a newly-developed eDNA detection system to its intra-genetic variation and phylogeographic breaks. As a result, several lineages of B. toni were identified, which presumably reflects historical migratory patterns of this species in Hokkaido. This study illustrates effectiveness of the eDNA technique to characterize not only species distribution trends but also intra-genetic variations and phylogeographic patterns. With further analytical optimizations, this approach has the potential to advance phylogeographic studies for understanding historical diversification processes of the primary freshwater fishes and other various organisms without catching individuals.