We identified in-stream and off-stream characteristics that influenced various species diversity metrics in reaches of the Duck River Basin, Tennessee, USA. This relatively small basin is home to one of the most diverse freshwater fish faunas in North America. In all, over 325,000 native fish representing 136 native fish species were electrofished in 207 collections across 86 stations. Diversity of native species and of seven taxa and functional guilds, including imperiled species, increased with size of the catchment above a station, an effect that was mediated by altitude. After removing the effects of catchment and altitude, diversity was influenced by in-stream factors and mostly by off-stream land composition such as wetlands within 0.1 km from the channel and urban/suburban development within 2.5 km. Pastures next to streams unexpectedly increased diversity. Our analyses suggest that there are detectable hotspots associated with off-stream landscape characteristics where conservation efforts may be focused. Our results may encourage conservation planners to apply geospatial analyses to identify diversity hotspots based on land cover distributions and develop recommendations for management of specific stream reaches. Alternatively, geospatial analysis may be used by planners to estimate the impacts of alternative land-use scenarios, thus preemptively conserving species richness.