Native amphibians in Hokkaido 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 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 Bufo-specific environmental DNA (eDNA) detection system for understanding current distributions of this toad along Ishikari river system in Hokkaido. We detected their DNA from 32 out of 73 water samples from the river system. They include sites without any previous record of the invasion of this toad. Applying our data to an occupancy model, we found high eDNA capture probabilities when the toads were observed within 500-1000m. These results suggest that our detection system is useful for assessing the front line of their spreads and understanding the patterns of their invasions.