<p>Picocyanobacteria (Pcy) are small (0.2–2 µm) photosynthetic prokaryotes commonly found in the oceans and freshwaters worldwide and are important primary producers of biomass in both oligotrophic and eutrophic waters. Eutrophication and subsequent seasonal blooms of harmful cyanobacteria (Hcy), predominantly Microcystis, are usual during high-temperature seasons in the Daechung Reservoir, in Korea. Therefore, much effort has been put into monitoring and improving the water quality of the Daechung Reservoir, with a major focus on Hcy. However, little attention has been paid to Pcy, partly because the cell identification and enumeration methods are unable to detect them. Herein, the appearance patterns of Pcy and Hcy are identified via next-generation sequencing and flow cytometry. The results indicate that Cyanobium dominates the upstream regions of Daechung Lake from June to July (Dominance ratios: 33.3 ~ 94.2%) and is succeeded by Microcystis from August to September (Dominance ratios: 38.6 ~ 89.7%). However, Microcystis is less evident in middle and downstream locations, and Cyanobium continues to dominate even in summer (average dominance ratio: 66.1%). Quantitative results indicate two periods (June–August and September–October) of high occurrences of Pcy at the upstream points (average cell density: 11,639 cells/mL), with Pcy and bloom-forming cyanobacteria increasing simultaneously in the summer. In the middle and downstream sites, however, the cell densities of Pcy are relatively low throughout the year (annual average cell density: 3451 cells/mL). High correlations are indicated between Pcy, water temperature, pH, and N-source nutrients, while Microcystis shows high positive correlations with phycocyanin concentration and P-sources nutrients. Therefore, a correlation analysis could be used to predict future blooms based on the appearance characteristics of Pcy and Hcy in freshwater ecosystems.</p>