The significance of archaea in regulating biogeochemical processes has led to an interest in their community compositions. Using 454 pyrosequencing, the present study examined the archaeal communities along a subtropical estuary, Pearl Estuary, China. Marine Group I Thaumarchaeota (MG‐I) were predominant in freshwater sites and one novel subgroup of MG‐I, that is MG‐Iν, was proposed. In addition, the previously defined MG‐Iα II was grouped into two clusters (MG‐Iα II‐1, II‐2). MG‐Iα II‐1 and MG‐Iλ II were both freshwater‐specific, with MG‐Iα II‐1 being prevalent in the oxic water and MG‐Iλ II in the hypoxic water. Salinity, dissolved oxygen, nutrients and pH were the most important determinants that shaped the differential distribution of MG‐I subgroups along Pearl Estuary. Marine Group II Euryarchaeota (MG‐II) dominated the saltwater sites, but their abundance was higher in surface waters. The phylogenetic patterns of MG‐I subgroups and their habitat preferences provide insight into their phylogeographic relationships. These results highlight the diversification of various ecotypes of archaea, especially of MG‐I, under distinct environmental factors in Pearl Estuary, which are of great value for further exploring their ecological functions.