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We studied the size-fractionized phytoplankton community structure characteristics and their response to environmental factors by investigating the photosynthetic pigment concentration and composition; composition and abundance of algal phyla levels; and environmental factors in typical lakes of Hubei Southeastern, China for four seasons. High-performance liquid chromatography (HLPC) results show that the total chlorophyll concentration was 36,418.62 mg/m³, and the contribution rates of microplankton, nanoplankton, and picoplankton to the total biomass were 13.43%, 49.08%, and 37.49%, respectively. Fucoxanthin, alloxanthin, zeaxanthin, and chlorophyll b were the main photosynthetic pigments. However, the spatial and temporal distribution had significant differences in the four typical lakes. Chemical taxonomy (CHEMTAX) calculation indicates that the dominant species were Diatoms and Cryptophytes in spring, Euglenophytes in summer, Euglenophytes and Cyanobacteria in autumn, and Euglenophytes and Cryptophytes in winter. The Chrysophyte and Dinoflagellates have the lowest proportion for all seasons. The redundancy analysis (RDA) demonstrates that the key environmental factors for the succession were Total Nitrogen (TN) and Total Phosphorus (TP). The application of the HPLC-CHEMTAX method has provided the first analysis of the community structure of size-fractionized phytoplankton in typical lakes of Hubei Southeast, China, and environmental factors affect the succession of size-fractionized phytoplankton over time. This study provides theoretical bases that the comprehensive research on different size phytoplankton in freshwater. [ABSTRACT FROM AUTHOR]