Alteration of rivers for human use has resulted in substantial biodiversity declines, particularly for species restricted to the largest rivers. Conservation and restoration efforts on large rivers often focus on the mainstem, but societal reliance on benefits derived from these alterations generally prevents complete restoration of the river. We propose that certain tributaries, by virtue of their lower degree of alteration, offer underappreciated opportunities for conserving large-river biota. Using the distribution patterns of large-river specialist fishes from the Mississippi River Basin, we identify a threshold discharge (166 cubic meters per second) beyond which tributaries support all or most of these species. We merge our macroecological analysis of assemblage structure with data on dam locations to identify tributaries where restoration efforts offer the highest potential conservation gains for 60 of the 68 large-river specialist fish species that are of state, federal, or international conservation concern. Given the highly altered state of many mainstem rivers, this analytical approach could be used to select tributaries that will aid in the conservation of large-river species worldwide.