There is a growing interest in interactions between spring-fed and runoff rivers for conserving the health of river ecosystems. In particular, the stable temperature regime and slow current velocities in spring-fed streams are expected to provide refuges for fishes from flow and temperature extremes. However, how fishes seasonally utilize combinations of spring-fed and runoff rivers as habitat remains poorly known. Here, we investigated seasonal changes in population density and muscle carbon and nitrogen stable isotope ratios of juvenile masu salmon (Oncorhynchus masou) in spring-fed tributaries and adjacent runoff mainstems in the Shubuto River system, northern Japan. We also collected benthic macroinvertebrates as potential prey items for fish at the study site. The biomass of benthic macroinvertebrates was one-order of magnitude greater in spring-fed tributaries than in runoff rivers throughout the season, associated with higher fine sediment cover and greater standing stock of detritus on the streambed. Juvenile masu salmon were especially aggregated in the spring-fed tributaries in winter compared to other seasons. Among seasons, the stable isotope analysis revealed that individuals that presumably had recently inhabited mainstems were most frequently caught in spring-fed tributaries in February. These results suggest that warmer and slower current velocities of spring-fed tributaries can provide attractive wintering habitat for juvenile masu salmon. Our findings highlight the important ecosystem functions of spring-fed tributaries in structuring animal communities within river ecosystems.