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Aim Streamflow and water temperature are primary variables influencing the distribution of freshwater taxa. Climate-induced changes in these variables are already causing shifts in species distributions, with continued changes projected in the coming decades. The Mobile River Basin (MRB), located in the southeastern United States, contains some of the highest levels of temperate freshwater biodiversity in North America. We integrated species distribution data with contemporary and future streamflow and water temperature data as well as other physical habitat data to characterize occurrence probabilities of fish species in the MRB with the goal of identifying current and future areas of high conservation value. Location Mobile River Basin, southeastern United States. Methods We used a maximum entropy approach to estimate baseline and future occurrence probability distributions for 88 fish species in the MRB based on model-generated streamflow and water temperature as well as geologic, topographic and land cover data. Areas of conservation prioritization were identified based on regions that contain suitable habitat for high levels of biodiversity according to baseline and future conditions while accounting for uncertainty associated with multiple future climate projections. Results On average, flow (28%), water temperature (28%) and geology (30%) contribute evenly to determining suitable habitat for fish species in the MRB. Based on baseline and future species distribution model estimates, high priority streams (best 10%) are largely concentrated in the eastern portion of the MRB, with a majority (51%) located within the Coosa and Tallapoosa River systems. Main conclusion We provide a framework that uses relevant hydrologic and environmental data in the context of future climatic uncertainty to estimate areas of freshwater conservation opportunity in the coming decades. While streamflow and water temperature represent important habitat for freshwater fishes in the MRB, distributions are also constrained by other aspects of the physical environment.