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ABSTRACT Nitrous oxide (N₂O) reductase, the sole natural microbial sink for N₂O, exists in two microbial clades:nosZI andnosZII. Although previous studies have explored inter‐clade ecological differentiation, the intra‐clade variations and their implications for N₂O dynamics remain understudied. This study investigated both inter‐ and intra‐clade ecological differentiation among N₂O reducers, the drivers influencing these patterns, and their effects on N₂O emissions across continental‐scale river systems. The results showed that bothnosZI andnosZII community turnovers were associated with similar key environmental factors, particularly total phosphorus (TP), but these variables explained a larger proportion of variation in thenosZI community. The influence of mean annual temperature (MAT) on community composition increased for more widespread N₂O‐reducing taxa. We identified distinct ecological clusters within each clade of N₂O reducers and observed identical ecological clustering patterns across both clades. These clusters were primarily characterized by distinct MAT regimes, coarse sediment texture as well as low TP levels, and high abundance of N₂O producers, with MAT‐related clusters constituting predominant proportions. Intra‐clade ecological differentiation was a crucial predictor of N₂O flux and reduction efficiency. Although different ecological clusters showed varying or even contrasting associations with N₂O dynamics, the shared ecological clusters across clades exhibited similar trends. Low‐MAT clusters in both thenosZI andnosZII communities were negatively correlated with denitrification‐normalized N₂O flux and the N₂O:(N₂O + N₂) ratio, whereas high‐MAT clusters showed positive correlations. This contrasting pattern likely stems from low‐MAT clusters being better adapted to eutrophic conditions and their more frequent co‐occurrence with N₂O‐producing genes. These findings advance our understanding of the distribution and ecological functions of N₂O reducers in natural ecosystems, suggesting that warming rivers may have decreased N₂O reduction efficiency and thereby amplify temperature‐driven emissions.