Understanding the upstream and downstream effect of impoundments on stream fish assemblages is important in managing fish populations and predicting the effects of future human activities on stream ecosystems. We used information collected over a 41-year period (1960-2001) to assess changes in fish assemblage structure resulting from impoundment of the Laramie River by Grayrocks Reservoir. Prior to impoundment (i.e., 1960-1979), fish assemblages were dominated by native catostomids and cyprinids. After impoundment several exotic species (e.g., smallmouth bass [Micropterus dolomieu], walleye [Sander vitreus; formerly Stizostedion vitreum], yellow perch [Perca flavescens], brown trout [Salmo trutta]) were sampled from reaches upstream and downstream of the reservoir. Suckermouth minnows (Phenacobius mirabilis) were apparently extirpated, and hornyhead chubs (Nocomis biguttatus) and common shiners (Luxilus cornutus) became rare upstream of Grayrocks Reservoir. The lower Laramie River downstream from Grayrocks Reservoir near its mouth retains habitat characteristics similar to those prior to impoundment (e.g., shallow, braided channel morphology) and is the only downstream area where several sensitive species persist, including sucker-mouth minnows, hornyhead chubs, and bigmouth shiners (Notropis dorsalis). Grayrocks Reservoir serves as a source of exotic piscivores to both upstream and downstream reaches and has altered downstream habitat characteristics. These impacts have had a substantial influence on native fish assemblages. Our results suggest that upstream and downstream effects of impoundment on fish assemblage structure are similar and that downstream reaches which retain habitat characteristics similar to pre-impoundment conditions may serve as areas of refuge for native species.