A total of 71 stream sites representing a gradient of urban land use was sampled across the Piedmont of the southeastern U.S. in 2014. Fish data collected (observed) at each site were compared to an expected community based on georeferenced historical (~1950 - ~1990) species occurrence records for stream segments (1:100,000 scale) containing the sampled stream sites. Loss of expected fish species (percent of fish species expected to occur but not observed) and homogenization (difference in Jaccard’s similarity of the fish community among sites observed and expected) were determined. On average, there was a 13.2% increase in the similarity of fish communities across sites, demonstrating evidence of community homogenization. Occurrence of Redbreast Sunfish (Lepomis auritus), Green Sunfish (L. cyanellus), and Bluegill (L. macrochirus) increased more than 50% over time (between observed and expected). Species loss increased significantly with urbanization whereas homogenization was not related to urbanization. Random forest analysis indicated that herbicides, insecticides, and centrarchid species richness were significant predictors of species loss. Of these, generalized additive model regression indicated that herbicides represented the most parsimonious model based on a single predictor. Stream base flow, elevation, and total nitrogen were significant predictors of homogenization. Generalized additive model regression indicated that decreased stream base flow was the single most important factor associated with increased homogenization. Chemical contaminants and associated ecosystem alteration and changes in stream flow may represent important regional influences on changes in fish communities in urban streams in the southeastern U.S.