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.