The Withlacoochee River in central Florida is a large blackwater river lacking substantive data on fish assemblage and has undergone extensive hydrological modification since the early 1900s. We compared fish assemblages in river segments above and below 2 major dams by conducting electrofishing surveys between April 2016 and June 2018. We also compiled historical records to assess temporal changes in species occurrence. We found that fish assemblages were significantly different in the lower segment than in the middle and upper segments. The lower segment had the lowest total catch-per-unit-distance and freshwater-fish species richness. Fish assemblage patterns were most highly correlated with Secchi depth, specific conductance, and percent of non-vegetative habitat cover. Much of the variation in fish assemblages among segments was consistent with that observed in undammed coastal rivers, but the lack of estuarine species in upper reaches of our study area suggests that the Inglis Dam may restrict upstream dispersal of these taxa. Comparison of species composition with historical records did not reveal any potential extirpations since 1931, but we recorded 8 marine species and 3 non-native species that were not present in historical records.