Humans have significantly impacted river systems. These impacts include long-term changes in the hydrological regimes of watersheds associated with changes in land use. Research shows that fish assemblages are modified in response to these changes. For this study, we examined whether fish assemblages were modified by anthropogenic land use. We hypothesized that fish assemblages with significant taxonomic and functional structure shifts would occur in watersheds where urban and agricultural land use are most prevalent. We analyzed species abundances and functional traits for 30 watersheds in the North Carolina region using temporal data from the RivFishTIME database. Principal components analyses followed by tests for correlation with year were conducted in each watershed using taxonomic and functional trait data of fish assemblages. We then used correlation analyses to test if temporal changes of taxonomic and functional structure of fish assemblages were correlated with land use classified as urban, agricultural, or undeveloped. We found few major shifts in taxonomic and functional structure of fish assemblages for available sampling periods. Relatively low urban and agricultural land use percentages do not result in significant fish assemblage response. Understanding temporal variation of fish assemblages in relation to land use is essential when managing conservation efforts of riverine systems.