Abstract. The niche differentiation of ammonia and nitrite oxidizers is controversial because they display disparate patterns in estuarine, coastal, and oceanic regimes. We analyzed diversity and abundance of ammonia-oxidizing archaea (AOA) and β-proteobacteria (AOB), nitrite-oxidizing bacteria (NOB), and nitrification rates to identify their niche differentiation along a salinity gradient from the Pearl River estuary to the South China Sea. AOA were generally more abundant than β-AOB; however, AOB more clearly attached to particles compared with AOA in the upper reaches of the Pearl River estuary. The NOB Nitrospira had higher abundances in the upper and middle reaches of the Pearl River estuary, while Nitrospina was dominant in the lower estuary. In addition, AOB and Nitrospira could be more active than AOA and Nitrospina since significantly positive correlations were observed between their gene abundance and the nitrification rate in the Pearl River estuary. There is a significant positive correlation between ammonia and nitrite oxidizer abundances in the hypoxic waters of the estuary, suggesting a possible coupling through metabolic interactions between them. Phylogenetic analysis further revealed that the AOA and NOB Nitrospina subgroups can be separated into different niches based on their adaptations to substrate levels. Water mass mixing is apparently crucial in regulating the distribution of nitrifiers from the estuary to open ocean. However, when eliminating water mass effect, the substrate availability and the nitrifiers' adaptations to substrate availability via their ecological strategies essentially determine their niche differentiation.