Abstract: morphology, ultrastructure, and 18S rRNA gene sequences of two strains of eukaryotic picoplankton isolated from Lake Dianchi, China, were analysed. Results showed that cellular shape, structure and autosporulation of two strains were similar to characteristics of the genus Mychonastes Simpson Van Valkenburg. The cell wall was consisted of double layers. The outer layer had a network of ribs on its surface and a trilaminar sheath, which appeared as a dark-light-dark triple layer. Cells had a minimal set of organelles, typical for small coccoid green algae, a single nucleus, one mitochondrion, one chloroplast without pyrenoid. Chloroplast was parietal, cup-shaped and occupied most of the cell volume. The phylogenetic tree based on 18S rRNA gene sequence data showed that isolated strains belonged to the genus Mychonastes, which belonged to the Chlorophyceae (Chlorophyta). Mychonastes was a firstly recorded genus of eukaryotic freshwater picoalgae in China.