Potamodromous Rainbow Trout Oncorhynchus mykiss are an important ecological and recreational resource in freshwater ecosystems of Alaska, and increased human development, hydroelectric projects, and reduced escapement of Chinook Salmon Oncorhynchus tshawytscha may threaten their populations. We used aerial and on-the-ground telemetry tracking, a digital landscape model, and resource selection functions to characterize seasonal movements and habitat use of 232 adult (>400 mm FL) Rainbow Trout across the complex, large (31,221 km²) Susitna River basin of south-central Alaska during 2003–2004 and 2013–2014. We found that fish overwintered in main-stem habitats near tributary mouths from November to April. After ice-out in May, fish ascended tributaries up to 51 km to spawn and afterward moved downstream to lower tributary reaches, assumedly to intercept egg and flesh subsidies provided by spawning salmonids in July and August. Fish transitioned back to main-stem overwintering habitats at the onset of autumn when salmonid spawning waned. Fidelity to tributaries where fish were initially tagged varied across seasons but was high (>0.75) in three out of four drainages. Model-averaged resource selection functions suggested that Rainbow Trout habitat use varied seasonally; fish selected low-gradient, sinuous, main-stem stream reaches in the winter, reaches with suitably sized substrate during spawning, larger reaches during the feeding season prior to the arrival of spawning salmonids, and reaches with high Chinook Salmon spawning habitat potential following the arrival of adult fish. We found little difference in movement patterns between males and females among a subset of fish for which sex was determined using genetic analysis. As most Rainbow Trout undertake extensive movements within and among tributaries and make use of a variety of seasonal habitats to complete their life histories, it will be critical to take a basinwide approach to their management (i.e., habitat protection and angling bag limits) in light of anticipated land-use changes.