Sixteen darter species, including the federally endangered Boulder Darter Etheostoma wapiti, are known to occur in the Elk River, a large, flow-regulated tributary of the Tennessee River, Tennessee–Alabama. Since the construction of Tims Ford Dam (TFD) in 1970, habitat modification caused by cold, hypolimnetic water releases and peak-demand hydropower generation has contributed to population declines and range reductions for numerous aquatic species in the main-stem Elk River. We developed Bayesian hierarchical multispecies occupancy models to determine the influence of site- and species-level characteristics on darter occurrence by using presence–absence data for 15 species collected from 39 study sites. Modeling results indicated that large-river obligate species, such as the Boulder Darter, were 6.92 times more likely to occur for every 37-km increase in the distance downstream from TFD. In contrast, small-stream species were 2.35 times less likely and cosmopolitan species were 1.88 times less likely to occur for every 37-km increase in distance downstream from TFD. The probability of occurrence for darter species also had a strong negative relationship with the absence of cobble and boulder substrates and the presence of high silt levels, particularly for species that require boulder substrates during spawning. Although total darter species richness was similar across all 39 sample sites, the composition of darter assemblages varied substantially among locations, presumably due in part to species-specific habitat affinities and hydrothermal conditions. The use of multispecies occupancy models allowed us to account for the incomplete detection of species while estimating the influence of physical habitat characteristics and species traits on darter occurrences, including rarely observed species that would have been difficult to model individually.