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Abstract. Nitrous oxide (N2O), a potent greenhouse gas and ozone-destroying agent, is produced during nitrogen transformations in both natural and human-constructed environments. Wastewater treatment plants (WWTPs) produce and emit N2O into the atmosphere during the nitrogen removal process. However, the impact of WWTPs on N2O emissions in downstream aquatic systems remains poorly constrained. By measuring N2O concentrations at a monthly resolution over a year in the Potomac River Estuary, a tributary of Chesapeake Bay in the eastern United States, we found a strong seasonal variation in N2O concentrations and fluxes: N2O concentrations were larger in fall and winter but the flux was larger in summer and fall. Observations at multiple stations across the Potomac River Estuary revealed hotspots of N2O emissions downstream of WWTPs. N2O concentrations were higher at stations downstream of WWTPs compared to other stations (median: 21.2 nM vs 16.2 nM) despite the similar concentration of dissolved inorganic nitrogen, suggesting the direct discharge of N2O from WWTPs into the aquatic system or a higher N2O production yield in waters influenced by WWTPs. Since wastewater production has increased substantially with the growing population and is projected to continue to rise, accurately accounting for N2O emissions downstream of the WWTPs would better constrain the global N2O emissions. Efficient N2O removal, in addition to dissolved nitrogen removal, should be an essential part of water quality control in WWTPs.