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Understanding the distribution of invasive species and their reproductive area is crucial for their managements after invasion. While catch and observation surveys are still embraced, environmental DNA (eDNA) has been increasingly utilized as an efficient tool for identifying these species in the wild. In this study, we developed a  Bufo -specific eDNA assay for detecting an invasive, toxic, and terrestrial toad species  Bufo japonicus formosus  in Hokkaido, Japan, and applied it to their reproductive area at watershed scale. The eDNA assay was field-validated in ponds where  B. japonicus  were observed, as well as in rivers downstream of the reproductive ponds. Thus, the assay provided us an opportunity to screen watersheds that include their reproductive area by collecting downstream water samples. Applying it to the Ishikari river basin, the largest river basin in Hokkaido (c.a., 14,330 km 2 ), we detected toad eDNA at 32 out of 73 sampling sites. They are composed of eleven sites with species observation records nearby (all the sites with observation records within a 500 m radius) and 21 sites without such records. And those eDNA detections were from twelve out of 31 river systems in the entire river basin. A Bayesian, multiscale occupancy model supported high eDNA detectability among those sites. These results suggest that the eDNA assay can efficiently estimate the presence of reproductive area of the terrestrial toad even from a distant downstream of the watershed, and that it provides a powerful means of detecting new reproductive area and monitoring further spread of invasive species.

Utilizing environmental DNA for wide-range distributions of reproductive area of an invasive terrestrial toad in Ishikari river basin in Japan