The southeastern United States is a global hotspot for freshwater biodiversity. There are at least 600 freshwater fish species found in river systems across the southeastern United States, of which about half are locally endemic with limited geographic range. However, following nearly two centuries of habitat modification, fragmentation, and land use change, many southeastern freshwater fish populations are now vulnerable to local extinction. We compiled stream fish sampling data for over 18,000 sites across the southeastern United States and generated lists of candidate species extirpations for watersheds with sufficient monitoring data at the HUC-8 watershed scale. During a workshop held in 2023, the candidate lists were reviewed by a group of experts from across the Southeast with deep knowledge of freshwater fish distributions and assigned an extirpation status to candidate species in 134 watersheds. Over half of the watersheds assessed (n = 78) are hypothesized to have lost at least one species (range: 1 – 17 species extirpated) and 113 species are presumed extirpated from at least one HUC-8 watershed (range: 1 – 10 watersheds). We fit hurdle models to examine how land use change, habitat fragmentation, and watershed characteristics are associated with the variation in the presence of any extirpations and the number of expected extirpations in HUC-8 watersheds. This analysis can be used to identify landscape features associated with “extirpation hotspots” and watersheds where additional extirpations are likely absent appropriate conservation actions. For example, our preliminary modeling identified fifteen watersheds that may have at least one extirpated species but currently have no documented extirpations. The ability to infer where there may be undetected extirpations and the compilation of a publicly available database of documented fish extirpations can help target conservation, e.g., to watersheds that support species that have been extirpated elsewhere and locations that could benefit from reintroduction efforts.