Native amphibians in Hokkaido (Rana pirica and Hynobius retardatus) are facing to the threats of alien toad (Bufo japonicus formosus) introduced from Honshu to Hokkaido about several decades ago. Because the alien toad has toxic substances (Bufotoxin), the native amphibian larvae easily die after consumption of the toad hatchlings. While grasping current distribution of alien species is crucial for planning of extermination and for protection of native amphibians, the concealing habit in terrestrial stage of B. japonicus makes it difficult to estimate. In our study, we evaluated applicability of a newly developed B. japonicus species-specific environmental DNA detection system for understanding current distributions of this toad along Ishikari river system in Hokkaido. We detected their DNA from 45 out of 93 water samples from various environments in the river system (swamps, ponds, branches and mainstream) including the sites without any previous record of the invasion of this toad. Notably, they included 31 samples from the river branches, by which the toad invasion into each branch can be specified. These results suggest that our detection system is useful for assessing the front line of their spreads and understanding the patterns of their invasions.