Three cruises were carried out in the Yangtze Estuary and its adjacent areas in May, November, June during 2009-2010. The spatial variations of phytoplankton community structure were investigated based on RP-HPLC analysis of pigments and CHEMTAX processing of the pigment data. 21 kinds of pigments were detected, among which chlorophyll a, peridinin, fucoxanthin, 19'-butanoyloxyfucoxanthin, 19'-hexanoyloxyfucoxanthin, chlorophyll b, diadinoxanthin, alloxanthin and zeaxanthin were the major pigments in the Yangtze Estuary and its adjacent areas. Chlorophyll a was the most abundant in all pigments, followed by fuxoxanthin. Other pigments generally contributed a minor proportion to the total pigments. High concentrations of fucoxanthin and peridinin were observed in May 2009 and June 2010, indicating blooms of diatoms and dinoflagellates. The results showed that the composition and distribution of phytoplankton pigments were influenced by environmental factors. The phytoplankton community, as determined by biomarker pigment concentration using HPLC and CHEMTAX, was composed mainly of diatoms, dinoflagellates, cryptophytes, chlorophytes, cyanobacteria, prymnesiophytes, chrysophytes and prasinophytes. The dominant algal groups were diatoms, dinoflagellates and chlorophytes in May 2009. The phytoplankton community was characterized by high contribution of diatoms in November 2009. Diatoms, dinoflagellates and cryptophytes accounted for 62.5% of chlorophyll a in June 2010, and the relative abundance of cyanobacteria was higher in this cruise. The spatial variations of phytoplankton community structure featured distinct regionality. Diatoms, chlorophytes and cryptophytes were the main groups in the inshore waters, and the abundances of prymnesiophytes, chrysophytes and cyanobacteria were increasing from inshore to the open sea.