Serial impoundment of major rivers leads to alterations of natural flow dynamics and disrupts longitudinal connectivity. Catostomid fishes (suckers, family Catostomidae) are typically found in riverine or backwater habitats yet are able to persist in impounded river systems. To the detriment of conservation, there is limited information about distribution of catostomid fishes in impounded rivers. We examined the longitudinal distribution of catostomid fishes over 23 reservoirs of the Tennessee River reservoir cascade, encompassing approximately 1600 km. Our goal was to develop a basin-scale perspective to guide conservation efforts. Catostomid species composition and assemblage structure changed longitudinally along the reservoir cascade. Catostomid species biodiversity was greatest in reservoirs lower in the cascade. Assemblage composition shifted from dominance by spotted sucker Minytrema melanops and buffalos Ictiobus spp. in the lower reservoirs to carpsuckers Carpiodes spp. midway through the cascade and redhorses Moxostoma spp. in the upper reservoirs. Most species did not extend the length of the cascade, and some species were rare, found in low numbers and in few reservoirs. The observed gradients in catostomid assemblages suggest the need for basin-scale conservation measures focusing on three broad areas: (1) conservation and management of the up-lake riverine reaches of the lower reservoirs, (2) maintenance of the access to quality habitat in tributaries to the upper reservoirs and (3) reintroductions into currently unoccupied habitat within species' historic distributions