Phylogenetic diversity of Synechococcus with different pigmentation in subtropical estuarine and coastal waters of Hong Kong was revealed by the phylogeny of cpcBA and cpeBA operons encoding for phycocyanin (PC) and phycoerythrin (PE). Synechococcus containing only PC (PC-rich Synechococcus) dominated at the estuarine station in summer, whereas PE-rich marine Synechococcus containing both PC and PE (PE-rich Synechococcus) dominated in the coastal waters. Our PC sequences are closely related to freshwater strains but differed from Baltic Sea strains, implying that they were from river discharge. Among PE-rich Synechococcus, clones grouping with strains containing only phycoerythrobilin (PEB-only) were abundant in July, while clones grouping with strains possessing a low content of phycourobilin (PUB) in addition to PEB (low PUB/PEB) were more abundant in January at both stations. Clones of high PUB/PEB types were only presented at the coastal station, but were not detected at the estuarine station. The much higher diversity of both PC-rich and PE-rich Synechococcus, as compared with the Baltic Sea, and the occurrence of the high PUB/PEB strains indicate the high dynamic nature of this subtropical estuarine-coastal environment with strong mixing of water masses ranging from Pearl River plume to oceanic South China Sea water. Our results of phylogenetic study agreed well with flow cytometric counts, which revealed the coexistence of PC-rich and PE-rich Synechococcus in the subtropical coastal waters and the dominance of the former type in the estuarine waters during summer high freshwater discharge. These results indicate that picocyanobacteria, particularly PC-rich Synechococcus, which has long been overlooked, are an important part of the primary production, and they could play an important role in the microbial food web in estuarine ecosystems.