We investigated variation in environmental conditions and fish assemblages over space and time in the upper Red River drainage (Oklahoma–Texas) using samples from eight sites (three tributaries and five mainstems) collected monthly in 1989–1990 and again in 1998–1999. Fish assemblages varied spatially, with geographically ubiquitous and ecologically tolerant taxa dominating tributaries and obligate species of prairie minnows common in mainstems. At the regional scale, fish assemblages were predictable between time periods. This resulted from strong longitudinal structuring in fishes across sites, which likely was influenced by spatial variation in environmental conditions across the drainage. These patterns suggested that at the regional scale strong forcing of fish by their environment influenced predictability of fishes between time periods. At the local scale, predictability in fish assemblages between time periods differed across type of habitat. Specifically, assemblages in tributary sites varied randomly over time, whereas mainstem assemblages were predictable. We believe that local environmental conditions favored different suites of taxa in tributaries between time periods. When we compared variation in fish assemblages to variation in environmental conditions at both regional and local scales, we detected that structure of fish assemblages tracked environmental variability at the regional scale. However, at the local scale, fish assemblages did not track environmental conditions. This suggests that environmental variation may affect fishes in a relative sense across the drainage, whereas, within sites, fishes may be decoupled from environmental structuring-forces. Based on the predictable patterns in fishes within the Red River drainage, we propose that monitoring efforts, aimed at detecting human-induced changes in this system, should include a range of mainstem sites that capture the strong structuring forces that vary longitudinally within this river system.