Redband Trout Oncorhynchus mykiss gairdneri is a species of high conservation concern in the Kootenai River basin, United States and Canada. However, identifying the specific mechanisms influencing Redband Trout is difficult as its life history structure is largely unknown. Using otolith microchemistry analysis, we found that 18.5% (n = 66) of the fish sampled (n = 329) were a product of mainstem Kootenai River spawning. The remainder were fish (n = 264) that originated from one of 31 tributaries throughout the basin. Redband Trout were captured between 0 and 153 km from their assigned natal tributaries. Most migratory fish originated from tributaries in the uppermost portion of the study area and had migratory mothers. However, a diversity of life history types (e.g., multi-year resident, migratory) was observed in the population. Redband Trout were estimated to outmigrate from natal habitats at a variety of ages (0–3 years) and lengths (12–430 mm). Our results highlight diversity in the population structure of Redband Trout and provide information valuable to conservation efforts in the Kootenai River system. Interestingly, several tributaries identified as natal habitats of Redband Trout contain substantial obstacles originally considered barriers to upstream fish passage. In addition, some streams in the lower portion of the Kootenai River basin with degraded habitat that were not expected to contribute Redband Trout had natal assignments. These observations indicate previously unknown sources of Redband Trout and support the notion that improved fish passage in disconnected and degraded streams could increase production of Redband Trout throughout the Kootenai River basin. Restoration efforts in the mainstem that include considerations for spawning and natal habitat may also aid in conservation efforts for Redband Trout. Our research demonstrates the value of microchemistry in planning and evaluating habitat restoration to recover fishes in large rivers.