To examine the phytoplankton assemblages and the effect of diluted waters on them, a research cruise was conducted from July 19 to August 7, 2015 in the Zhujiang (Pearl) River estuary in the northern South China Sea (21°N–23.5°N, 111°E–117°E). Samples were collected from 65 stations including one for time-series sampling. A total of 212 phytoplankton taxa were identified from 61 genera belonging to 4 phyla. Among them, 122 species identified from 42 genera of Bacillariophyta and 83 species from 15 genera of Pyrrophyta. Chain-forming diatoms dominated the phytoplankton community where Pseudo-nitzschia delicatissima, Guinardia striata, Thalassionema nitzschioides, and P. pungens comprised about 52% of the total abundance. However, higher cell abundances concentrated on both sides of the estuary, because of low salinity and high nutrients brought by diluted water. In addition, Canonical Correspondence Analysis revealed that salinity and dissolved inorganic nitrogen shaped the species composition in the study area. Furthermore, the Jaccard similarity index showed prevailing high similarity in the distribution of species in low-salinity diluted waters, and the Bray-Curtis similarity depicted distinguished grouping for phytoplankton assemblages along the salinity gradient. However, phytoplankton diel vertical cycles showed maximum abundance occurred at 2?00 am, which was mainly contributed by benthic phytoplankton species Pseudo-nitzschia spp. and T . nitzschioides.