Assessing potential effects of flow regulation on southeastern warmwater fish assemblages is problematic because of high species richness and poor knowledge of habitat requirements for most species. Our study investigated relationships between availability and temporal persistence of key habitats and fish assemblage structure at regulated and unregulated sites in the Tallapoosa River system. Fish assemblage characteristics at seven sites were quantified based on 1,400 electrofishing samples collected during 1994 and 1995. Physical Habitat Simulation (PHABSIM) programs were used to model availability and persistence of key habitats at regulated and unregulated sites. Associations between fish assemblages and availability or persistence of key habitats were identified via regression and analysis of variance. We found that hydropeaking dam operation reduced the average length of time that shallow‐water habitats persisted and also reduced year‐to‐year variation in the persistence of shallow‐water habitats compared with unregulated sites. Across sites and years, proportional representation of catostomids was positively correlated with persistence of shallow and slow‐water habitats during spring. Proportion of individuals as cyprinids was positively correlated with median availability of deep–fast habitat whereas proportion of percids was inversely related to median availability of deep–fast habitat. Mean fish density was positively correlated with the persistence of shallow and slow‐water habitats. Comparisons of key‐habitat measures and fish abundances between 1994 and 1995 at each site indicated that higher abundances of percids, catostomids, and cyprinids were associated with increased availability and persistence of shallow and slow‐water habitats in 1995. These findings demonstrate that the temporal and spatial availability of key habitats could serve as useful measures of the potential effects of flow alteration on lotic fish assemblages and suggest that both short‐term persistence of key habitats as well as annual variation in key‐habitat availability are important for maintaining diverse fish assemblages.