<p>Isoscapes, which map spatial gradients in isotope ratios, can be used to estimate the life history of organisms. Comparisons of isoscape of strontium isotope ratios (⁸⁷Sr/⁸⁶Sr) in rivers with otolith isotope ratios can provide useful data for estimating the life history of fishes, such as their natal origin, growth habitats, and movement history. While several studies have constructed ⁸⁷Sr/⁸⁶Sr isoscapes at large spatial scales, no study has created ⁸⁷Sr/⁸⁶Sr isoscapes of river water at large spatial scales in Japan, such as entire river basins. In this study, we constructed a high-resolution ⁸⁷Sr/⁸⁶Sr isoscape for the Nagara River, which is known for producing valuable fishery resources, such as ayu (<i>Plecoglossus altivelis</i>) and satsuki-masu salmon (<i>Oncorhynchus masou</i> <i>ishikawae</i>). We collected water samples at 115 points across the Nagara River Basin and analyzed the ⁸⁷Sr/⁸⁶Sr of these samples. Then, we constructed an ⁸⁷Sr/⁸⁶Sr isoscape of the Nagara River basin based on the ⁸⁷Sr/⁸⁶Sr of the water samples and the geographical information of the basin. The isotope ratios of river water varied mainly because of the bedrock composition in the catchment area, with significant variations between tributaries and between the upstream and the downstream reaches of the river. This isoscape is considered useful for estimating the life history of fishes in the Nagara River basin and the impact of environmental change on the river’s fisheries resources.</p>