Wyoming’s Powder River is considered an example of a pristine prairie river system. While the river hosts a largely native fish assemblage and remains unimpounded over its 1,146-km course to the Yellowstone River confluence, the hydrologic regime has been altered through water diversion for agriculture and natural gas extraction and there has been limited study of fish assemblage structure. We analyzed fish data collected from the mainstem Powder River in Wyoming between 1896 and 2008. Shifts in presence/absence and relative abundance of fish species, as well as fish assemblage composition, were assessed among historical and recent samples. The recent Powder River fish assemblage was characterized by increased relative abundances of sand shiner Notropis stramineus and plains killifish Fundulus zebrinus, and decreases in sturgeon chub Macrhybopsis gelida. Shifts in fish species relative abundance are linked to their reproductive ecology with species with adhesive eggs generally increasing in relative abundance while those with buoyant drifting eggs are decreasing. Assemblage shifts could be the result of landscape level changes, such as the loss of extreme high and low flow events and changing land use practices.