Conserving biological resources native to large river systems increasingly depends on how flow-regulated segments of these rivers are managed. Improving management will require a better understanding of linkages between river biota and temporal variability of flow and instream habitat. However, few studies have quantified responses of native fish populations to multiyear (>2 yr) patterns of hydrologic or habitat variability in flow-regulated systems. To provide these data, we quantified young-of-year (YOY) fish abundance during four years in relation to hydrologic and habitat variability in two segments of the Tallapoosa River in the southeastern United States. One segment had an unregulated flow regime, whereas the other was flow-regulated by a peak-load generating hydropower dam. We sampled fishes annually and explored how continuously recorded flow data and physical habitat simulation models (PHABSIM) for spring (April-June) and summer (July-August) preceding each sample explained fish abundances. Patterns of YOY abundance in relation to habitat availability (median area) and habitat persistence (longest period with habitat area continuously above the long-term median area) differed between unregulated and flow-regulated sites. At the unregulated site, YOY abundances were most frequently correlated with availability of shallow-slow habitat in summer (10 species) and persistence of shallow-slow and shallow-fast habitat in spring (nine species). Additionally, abundances were negatively correlated with 1-h maximum flow in summer (five species). At the flow-regulated site, YOY abundances were more frequently correlated with persistence of shallow-water habitats (four species in spring; six species in summer) than with habitat availability or magnitude of flow extremes. The associations of YOY with habitat persistence at the flow-regulated site corresponded to the effects of flow regulation on habitat patterns. Flow regulation reduced median flows during spring and summer, which resulted in median availability of shallow-water habitats comparable to the unregulated site. However, habitat persistence was severely reduced by flow fluctuations resulting from pulsed water releases for peak-load power generation. Habitat persistence, comparable to levels in the unregulated site, only occurred during summer when low rainfall or other factors occasionally curtailed power generation. As a consequence, summer-spawning species numerically dominated the fish assemblage at the flow-regulated site; five of six spring-spawning species occurring at both study sites were significantly less abundant at the flow-regulated site. Persistence of native fishes in flow-regulated systems depends, in part, on the seasonal occurrence of stable habitat conditions that facilitate reproduction and YOY survival.