The Three Gorges Reservoir of China is a remarkable water conservancy project. It is thus significant to study the dynamics of the reservoir ecological system and plankton distribution. In this study, we used Sanger-based sequencing of the clone libraries of the 185 rRNA gene to analyze the picoeukaryotic diversity (size class 0.7-5 mu m) from two sampling sites along the Three Gorges Reservoir during the four seasons in 2013. The community investigated by our approach was dominated by three main phyla, Cryptophyta (30.88% of clones), Chlorophyta (9.6%), and Bacillariophyta (8.7%). In spring, the parasitic population of chytrids in the reservoir is high and may limit the abundance of diatoms. Moreover, the spatial pattern prevails over temporal variability in picoeukaryotic community structure of the Three Gorges Reservoir. Further analyses show that the spatial differences in community structure are mainly caused by the variability of non-dominant phyla. This leads to the hypothesis that the injection of tributaries will have a certain effect on the plankton community structure of the main stream of the Three Gorges Reservoir.