In Lake Biwa, picocyanobacteria blooms usually occur during the summer–autumn thermal stratification period. Intriguingly, a novel bloom was detected in winter 2015–2016, in which picocyanobacterial cell density increased by one order of magnitude despite lower water temperature, suggesting the possibility that “cold-water-preference” species dominate in the picocyanobacterial community. In the present study, we investigated the phylogenetic diversity of picocyanobacteria in Lake Biwa by analyzing the 16S rRNA gene. We found that the picocyanobacterial community were highly diverse in Lake Biwa, with eight Synechococcus-related operational taxonomic units (OTUs) detected in different seasons. These OTUs fell into distinct phylogenetic groups, and the majority were closely related to clusters reported previously. Notably, OTU04, detected during the winter bloom, was highly affiliated with sequences found in a variety of lakes, such as Tibetan lakes and Lake Superior, where the water bodies generally have a low trophic state and temperature, and different concentrations of total dissolved solids. Thus, we inferred that the group containing OTU04 may be a psychrotolerant lineage that is widely distributed in oligotrophic water systems with low–intermediate salinity.