Physical and hydrologic connectivity among river-reservoir ecosystem (RRE) segments, including the river, riverreservoir interface (RRI), and reservoir, requires consideration of management at larger spatial scales. Multiple objectives (e.g., ecosystem function, sport fisheries, and maximizing fish diversity) seem well suited to this broad spatial scale; however, data limitations regarding the distribution of taxa within RREs currently hamper such an approach. We quantified the fish assemblage of the Trinity River-Lake Livingston ecosystem in Texas, USA, using a stratified approach to determine the contribution of individual segments to the overall fish assemblage. In general, fish assemblages varied along a continuum from the river to the reservoir; however, fish assemblages in several river backwaters did not follow this gradient, thus adding diversity to the overall RRE. Variability in fish assemblages among ecosystem segments was the result of both species composition and relative abundance. Species richness (N = 58 total) was highest in the river segment (N = 49) followed by the RRI (N = 46) and was much lower in the reservoir (N = 36). In contrast, average catch per standardized unit of sampling effort (all species combined) was greatest in the RRI, about 25% lower in the reservoir, and about 50% lower in the river. Across the ecosystem, 26 species (45%) were considered common, while 32 species (55%) were considered rare. Notably, species of conservation need were not always most abundant in river habitats, and species supporting recreational fisheries were not always most dominant in the reservoir. These findings suggest that the RRE can serve as an appropriate spatial scale for both conservation and recreational fishery management actions.Regulation of rivers through impoundment provides many benefits to humans (e.g., water supply, electricity, and recreation); however, alteration of these ecosystems has been detrimental to many native fish species, especially fluvial specialists (e.g.,