Understanding the sources, transport, and spatiotemporal variability of contaminants of emerging concern (CECs) is important for understanding risks and developing monitoring and mitigation strategies. This study used mass balances to compare wastewater treatment plant (WWTP) and upstream sources of 16 CECs to a mixed-use watershed in Minnesota, under different seasonal and hydrological conditions. Three distinct CEC groups emerged with respect to their source proportionality and instream behavior. Agricultural herbicides and daidzein inputs were primarily via upstream routes with the greatest loadings and concentrations during high flows. Trimethoprim, mecoprop, nonprescription pharmaceuticals, and personal care products entered the system via balanced/mixed pathways with peak loadings and concentrations in high flows. Carbaryl, 4-nonylphenol, and the remaining prescription pharmaceuticals entered the system via WWTP effluent with relatively stable loadings across sampling events. Mass balance analysis based on multiple sampling events and sites facilitated CEC source comparisons and may therefore prove to be a powerful tool for apportioning sources and exploring mitigation strategies.