Plug-flow activated sludge reactors (ASR) that are step-feed with wastewater are widely adopted in wastewater treatment plants (WWTPs) due to their ability to maximise the use of the organic carbon in wastewater for denitrification. Nitrous oxide (N2O) emissions are expected to vary along these reactors due to pronounced spatial variations in both biomass and substrate concentrations. However, to date, no detailed studies have characterised the impact of the step-feed configuration on emission variability. Here we report on the results from a comprehensive online N2O monitoring campaign, which used multiple gas collection hoods to simultaneously measure emission along the length of a full-scale, step-fed, plug-flow ASR in Australia. The measured N2O fluxes exhibited strong spatial-temporal variation along the reactor path. The step-feed configuration had a substantial influence on the N2O emissions, where the N2O emission factors in sections following the first and second step feed were 0.68% ± 0.09% and 3.5% ± 0.49% of the nitrogen load applied to each section. The relatively high biomass-specific nitrogen loading rate in the second section of the reactor was most likely cause of the high emissions from this section.