1. There is growing international concern about declines in populations of anguillid eels, resulting in their inclusion in the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species. Monitoring the population dynamics of these species is often challenging, however, owing to their broad distributions and complex, catadromous life histories.2. Whether environmental DNA (eDNA) analysis could be used to monitor the spatial distribution of anguillid eels in rivers was investigated by conducting basin-scale surveys of Japanese eels, Anguilla japonica Temminck & Schlegel, 1846, in 10 rivers in Japan, and comparing the results obtained using eDNA analysis with the results obtained using the electrofishing method. Moreover, the relationship between the eDNA concentration and the abundance and biomass of Japanese eels was examined.3. The eDNA of Japanese eels was detected at 56 (91.8%) of the 61 study sites from which individuals were collected by electrofishing, and at an additional 35 sites where individuals were not directly collected. This indicates that eDNA analysis has a greater sensitivity for detecting the presence of eels, making it a powerful tool for monitoring the spatial distribution of anguillid eels in rivers. 4. A significant, but weak, positive relationship between the eDNA concentration and the abundance and biomass of Japanese eels was also found, suggesting that eDNA analysis may be useful for estimating the abundance and biomass of anguillid eels in rivers. 5. This is the first study to demonstrate the potential usefulness of eDNA analysis for estimating the spatial distribution, abundance, and biomass of Japanese eels in rivers. eDNA analysis will allow anguillid eel populations to be monitored over large spatial and temporal scales using a consistent protocol, with reduced time and effort compared with conventional techniques, providing invaluable information for managing populations of these endangered species.