Abstract: To systematically understand phytoplankton community structures and the corresponding impact factors, an investigation was carried out at 71 sites in the mainstream, eight primary tributaries, the Dongting Lake, and the Poyang Lake in the Yangtze River basin. The characteristics of the phytoplankton community structures were compared among different water areas and periods. The impact factors contributing to the differences of phytoplankton community structures were further explored. Results revealed that the abundance and biomass of phytoplankton in the Yangtze River basin ranged from 0.07×104 to 2 493.69×104 cells/L, and from 0.001 1 to 21.072 2 mg/L, respectively. Bacillariophyta, Chlorophyta, and Cyanophyta were the dominant phytoplankton phyla. From west to east of the Yangtze River basin, the total abundance and biomass of phytoplankton and proportion of Cyanophyta and Chlorophyta showed an increasing trend, while the proportion of Bacillariophyta abundance decreased gradually. The average total abundance and biomass of phytoplankton in the fish fattening period were higher than those in the fish breeding period and the overwintering period, characterized by higher proportion of Cyanophyta and Chlorophyta and lower proportion of Bacillariophyta. Moreover, the results of redundancy analysis, hierarchical partitioning, and variation partitioning indicated that both environmental and spatial variables contributed to the variance of phytoplankton community structures in the Yangtze River basin. In the fish fattening period, the environmental filtering effects were dominant, of which the water temperature was the most important factor and was strongly positively correlated with the total and dominant phyla abundance of phytoplankton. In the fish overwintering period, the differences in the phytoplankton community structure were better explained by spatial factors rather than the environmental factors, of which the spatial factor of MEM3 was the main factor. This study provided important data for the management of the aquatic ecology in the Yangtze River basin.