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Ammonia oxidation, the first and rate‐limiting step in nitrification, plays a critical role in the nitrogen cycle. However, the links between the dynamics of ammonia‐oxidizing communities and ecosystem processes along the estuarine salinity gradient remain uncertain. In this study, we examined the diversity, abundance, and community structure of ammonia‐oxidizing prokaryotes, and the potential nitrification rates along the Yangtze estuarine salinity gradient. Phylogenetic analysis showed that the predominant ammonia‐oxidizing bacteria (AOB) and ammonia‐oxidizing archaea (AOA) fell within the Nitrosospira and Nitrosopumilus clusters, respectively. The AOB amoA gene abundance (4.67 × 105 to 3.90 × 107 copies per gram of dry sediment) outnumbered AOA (5.14 × 104 to 8.88 × 106 copies per gram of dry sediment). The potential nitrification rates varied between 0.13 and 0.63 μg N·g−1·day−1 and related only to AOA amoA gene abundance. Salinity had significant effects on AOA amoA gene abundance, nitrification rates, and the community structure of ammonia‐oxidizing prokaryotes. Principal coordinate analysis showed that the AOB amoA gene clones derived from the middle‐ and high‐salinity regions behaved as a cohesive group, while all the low‐salinity clone libraries were grouped together. Moreover, the distribution of AOA communities showed a distinct salinity differentiation. Overall, this study improves the understanding of the dynamic shifts in ammonia‐oxidizing microorganisms in the Yangtze Estuary. Key Points: Significant shifts in ammonia‐oxidizing community structure were found along the salinity gradientBoth of AOA amoA gene abundance and potential nitrification rates were significantly and negatively correlated with salinityPotential nitrification rates were significantly related to AOA amoA gene abundance, but not with AOB [ABSTRACT FROM AUTHOR]