The hypothesis that temperate stream fishes alter habitat use in response to changing water temperature and stream discharge was evaluated over a 1 year period in the Neosho River, Kansas, U.S.A. at two spatial scales. Winter patterns differed from those of all other seasons, with shallower water used less frequently, and low-flow habitat more frequently, than at other times. Non-random habitat use was more frequent at the point scale (4.5 m2) than at the larger reach scale (20-40 m), although patterns at both scales were similar. Relative to available habitats, assemblages used shallower, swifter-flowing water as temperature increased, and shallower, slower-flowing water as river discharge increased. River discharge had a stronger effect on assemblage habitat use than water temperature. Proportion of juveniles in the assemblage did not have a significant effect. This study suggests that many riverine fishes shift habitats in response to changing environmental conditions, and supports, at the assemblage level, the paradigm of lotic fishes switching from shallower, high-velocity habitats in summer to deeper, low-velocity habitats in winter, and of using shallower, low-velocity habitats during periods of high discharge. Results also indicate that different species within temperate river fish assemblages show similar habitat use patterns at multiple scales in response to environmental gradients, but that non-random use of available habitats is more frequent at small scales. ?? 2006 The Fisheries Society of the British Isles.