The distribution of ichthyofauna in rivers with large environmental gradients and fragmented by riverine structures is determined by environmental suitability and habitat connectivity. In this study, we conducted an ichthyofauna survey in the Shinano River system (Chikuma River, Shinano River, and tributary rivers) from the headwaters to the river mouth by using environmental DNA. We sampled 15 sites from the mainstream of the 367-km-long Chikuma and Shinano Rivers and 15 sites from 15 tributary rivers. At each survey point, river water was collected according to a standard method, and environmental DNA was metabarcoded using MiFish primers for the comprehensive detection of freshwater fish. Here we report the patterns of fish diversity in different habitats, the relationship between ichthyofauna and riverine structures, the rare species detected, and the relationship between ayu release and the distribution of domestic invasive species.