During the past 100 years, most large rivers in North America have been altered for flood control, hydropower, navigation or water supply development. Although these activities clearly provide important human services, their associated environmental disturbances can profoundly affect stream-dwelling organisms. We used dynamic multi-species occupancy models combined with a trait-based approach to estimate the influence of site-level and species-level characteristics on patch dynamic rates for 15 darter species native to the Elk River, a large, flow-regulated Tennessee River tributary in Tennessee and Alabama. Dynamic occupancy modelling results indicated that for every 2.5 °C increase in stream temperature, darters were 3.94 times more likely to colonize previously unoccupied stream reaches. Additionally, large-bodied darter species were 3.72 times more likely to colonize stream reaches compared with small-bodied species, but crevice-spawning darter species were 5.24 times less likely to colonize previously unoccupied stream reaches. In contrast, darters were 2.21 times less likely to become locally extinct for every 2.5 °C increase in stream temperature, but high stream discharge conditions elevated the risk of local extinction. Lastly, the presence of populations in neighbouring upstream study reaches contributed to a lower risk of extinction, whereas the presence of populations in neighbouring downstream study reaches contributed to higher rates of colonization. Our study demonstrates the application of a trait-based approach combined with a metapopulation framework to assess the patch dynamics of darters in a regulated river. Results from our study will provide a baseline for evaluating the ecological consequences of alternative dam operations.