Longitudinal gradients of fish assemblage and habitat structure were investigated in the Kootenai River of northern Idaho. A total of 43 500-m river reaches was sampled repeatedly with several techniques (boat-mounted electrofishing, hoop nets and benthic trawls) in the summers of 2012 and 2013. Differences in habitat and fish assemblage structure were apparent along the longitudinal gradient of the Kootenai River. Habitat characteristics (e.g. depth, substrate composition and water velocity) were related to fish assemblage structure in three different geomorphic river sections. Upper river sections were characterized by native salmonids (e.g. mountain whitefish Prosopium williamsoni), whereas native cyprinids (peamouth Mylocheilus caurinus, northern pikeminnow Ptychocheilus oregonensis) and non-native fishes (pumpkinseed Lepomis gibbosus, yellow perch Perca flavescens) were common in the downstream section. Overall, a general pattern of species addition from upstream to downstream sections was discovered and is likely related to increased habitat complexity and additions of non-native species in downstream sections. Assemblage structure of the upper sections were similar, but were both dissimilar to the lower section of the Kootenai River. Species-specific hurdle regressions indicated the relationships among habitat characteristics and the predicted probability of occurrence and relative abundance varied by species. Understanding fish assemblage structure in relation to habitat could improve conservation efforts of rare fishes and improve management of coldwater river systems.