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<p>Nitrous oxide (<span class="inline-formula">N₂O</span>) has significant global warming potential as a greenhouse gas. Estuarine and coastal regimes are the major zones of <span class="inline-formula">N₂O</span> production in the marine system. However, knowledge on biological sources of <span class="inline-formula">N₂O</span> in estuarine ecosystems remains controversial but is of great importance for understanding global <span class="inline-formula">N₂O</span> emission patterns. Here, we measured concentrations and isotopic compositions of <span class="inline-formula">N₂O</span> as well as distributions of ammonia-oxidizing bacterial and archaeal <i>amoA</i> and denitrifier <i>nirS</i> genes by quantitative polymerase chain reaction along a salinity gradient in the Pearl River estuary, and we performed in situ incubation experiments to estimate <span class="inline-formula">N₂O</span> yields. Our results indicated that nitrification predominantly occurred, with significant <span class="inline-formula">N₂O</span> production during ammonia oxidation. In the hypoxic waters of the upper estuary, strong nitrification resulted in the observed maximum <span class="inline-formula">N₂O</span> and <span class="inline-formula">Δ</span><span class="inline-formula">N₂O</span><span class="inline-formula">_excess</span> concentrations, although minor denitrification might be concurrent at the site with the lowest dissolved oxygen. Ammonia-oxidizing <span class="inline-formula"><i>β</i></span>-proteobacteria (AOB) were significantly positively correlated with all <span class="inline-formula">N₂O</span>-related parameters, although their <i>amoA</i> gene abundances were distinctly lower than ammonia-oxidizing archaea (AOA) throughout the estuary. Furthermore, the <span class="inline-formula">N₂O</span> production rate and the <span class="inline-formula">N₂O</span> yield normalized to <i>amoA</i> gene copies or transcripts estimated a higher relative contribution of AOB to the <span class="inline-formula">N₂O</span> production in the upper estuary. Taken together, the in situ incubation experiments, <span class="inline-formula">N₂O</span> isotopic composition and concentrations, and gene datasets suggested that the high concentration of <span class="inline-formula">N₂O</span> (oversaturated) is mainly produced from strong nitrification by the relatively high abundance of AOB in the upper reaches and is the major source of <span class="inline-formula">N₂O</span> emitted to the atmosphere in the Pearl River estuary.</p>