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Nitrogen (N) pollution remains a crucial environmental problem in coastal waters affected by eutrophication globally, but the sources of the pollution are not often well quantified locally. This study explored how land use influences N concentrations and sources along 400 km of the Potomac River – the second largest tributary of the Chesapeake Bay located in the Mid-Atlantic United States. Our results showed that total dissolved nitrogen and nitrate (TDN and NO3−-N) in tributaries of the Potomac River were significantly correlated with watershed cropland percentage (R2 = 0.68, n = 31). As a result, TDN and NO3−-N concentrations along the Potomac River mainstem increased sharply from forest reaches to agricultural reaches followed by slowly decreasing downstream along urban reaches near Washington DC. NO3−-N and oxygen isotope ratios (δ15N–NO3- and δ18O–NO3-), both of which were highest in tributaries draining urban land use and lowest in tributaries draining forest land use, generally increased along the Potomac River mainstem across the forest-agricultural-urban land use gradient. Source tracking using δ15N–NO3- and δ18O–NO3- suggested that nitrate inputs from agriculture were the main sources in the major segments of the Potomac River, and inputs from forest soils and wastewater treatment plants were important in the headwater and the section below Washington D.C., respectively. TDN load estimations indicated >66% of TDN load of the Potomac River was from the agricultural zone, and >36% of TDN from agricultural and forested areas was retained within the urban river section during the summer low-streamflow period. This study highlights the importance of nonpoint agricultural sources to the Potomac River, as well as N retention within river channel. Given that some agricultural sources may persist further downriver even as urbanization increases, isotope source tracking can be used to prioritize more effective source reduction strategies in the Chesapeake Bay Watershed.