LAUSR

&NA; Lake Poyang, which is connected to the Yangtze River, is the largest freshwater lake in China and experiences large and frequent changes in water levels. The seasonal diversity and composition of photosynthetic picoeukaryotes (PPEs) in Lake Poyang were investigated from flow‐cytometry‐sorted samples using MiSeq high‐throughput sequencing. Flow cytometric counting indicated that PPEs accounted for 97% of the total picophytoplankton abundance in spring, reaching a maximum value of 6.30 × 104 cells mL‐1. PPEs in Lake Poyang showed lower diversity than those in other investigated lakes in the lower reaches of the Yangtze River and were dominated by one OTU (66.29%) affiliated with Mychonastes (Chlorophyceae). Other minor classes of PPEs were found to be sporadically abundant in specific seasons, i.e. Chrysophyceae prevailed in spring and summer, while Eustigmatophyceae was mainly present in winter. This study reports coccal green algae of Mychonastes in Lake Poyang; additionally, these algae are reportedly representative of the prominent plankton in this river‐connected lake ecosystem but are often overlooked due to their lack of morphological features. Finally, the sequencing results from the sorted samples of Lake Poyang revealed that the proportion of PPEs was quite low, with an average of 36% of total reads. Many OTUs belonging to heterotrophic picoeukaryotes were also identified in the sorted samples, most of which were affiliated with terrestrial fungi, including Basidiomycota and Ascomycota. The spores of these fungi can disperse in the aquatic environment during the flood seasons, yet their effect on PPEs is still unclear. &NA; Graphical Abstract Figure. Mychonastes dominates photosynthetic picoeukaryotes in a river‐connected lake.