Free-flowing river segments provide refuges for many imperiled aquatic biota that have been extirpated elsewhere in their native ranges. These biodiversity refuges are also foci of conservation concerns because species persisting within isolated habitat fragments may be particularly vulnerable to local environmental change. We have analyzed long-term (14- and 20-y) survey data to assess evidence of fish species declines in two southeastern U.S. rivers where managers and stakeholders have identified potentially detrimental impacts of current and future land uses. The Conasauga River (Georgia and Tennessee) and the Etowah River (Georgia) form free-flowing headwaters of the extensively dammed Coosa River system. These rivers are valued in part because they harbor multiple species of conservation concern, including three federally endangered and two federally threatened fishes. We used data sets comprising annual surveys for fish species at multiple, fixed sites located at river shoals to analyze occupancy dynamics and temporal changes in species richness. Our analyses incorporated repeated site-specific surveys in some years to estimate and account for incomplete species detection, and test for species-specific (rarity, mainstem-restriction) and year-specific (elevated frequencies of low- or high-flow days) covariates on occupancy dynamics. In the Conasauga River, analysis of 26 species at 13 sites showed evidence of temporal declines in colonization rates for nearly all taxa, accompanied by declining species richness. Four taxa (including one federally endangered species) had reduced occupancy across the Conasauga study sites, with three of these taxa apparently absent for at least the last 5 y of the study. In contrast, a similar fauna of 28 taxa at 10 sites in the Etowah River showed no trends in species persistence, colonization, or occupancy. None of the tested covariates showed strong effects on persistence or colonization rates in either river. Previous studies and observations identified contaminants, nutrient loading, or changes in benthic habitat as possible causes for fish species declines in the Conasauga River. Our analysis provides baseline information that could be used to assess effectiveness of future management actions in the Conasauga or Etowah rivers, and illustrates the use of dynamic occupancy models to evaluate evidence of faunal decline from time-series data.