To understand the spatio-temporal of phytoplankton communities and their key environmental drivers in the Lake Qinghai, we conducted a systematic survey during May (Spring), August (Summer), and October (Autumn) in 2020, and examined how phytoplankton communities varied among three seasons and four regions (lakeshore zone, inlet zone, shallow zone, and deep zone). A total of 65 species belonging to 39 genera and 6 phyla were identified, with Bacillariophyta (36 species, accounting for 55.38% of the total species) being the species-richest class, followed by Chlorophyta (17 species, 26.15%), and Cyanophyta (7 species, 10.76%). The abundance of phytoplankton differed significantly among four regions, with significantly higher values in deep zone than other zones, probably due to the more stable environment in deep zone. There were also significant differences in abundance and biomass of phytoplankton among the three seasons. The abundance and biomass of phytoplankton in summer and autumn were nearly 10 times higher than those in spring, and the dominant groups and species of phytoplankton also changed greatly. The most dominant class in Spring was Bacillariophyta, and the dominant species were also belonged to Bacillariophyta, while the dominant group and species in Summer and Autumn became Cyanophyta and its subset species. The Pielou's evenness and Simpson diversity indices of phytoplankton in Spring were significantly higher than those in Summer and Autumn, and the Margalef and Shannon-Wiener indices showed higher values in Autumn than in Summer. PERMANOVA and NMDS both indicated that the structure of phytoplankton communities varied significantly among different regions and seasons in the Lake Qinghai. In addition, the dbRDA analysis showed that salinity, water temperature and total phosphorus were the key environmental factors affecting the phytoplankton patterns in the Lake Qinghai. Compared with the historical surveys, this study indicated that the main dominant groups of phytoplankton community in Lake Qinghai gradually changed from Bacillariophyta to Cyanophyta. This study reveals the spatio-temporal pattern and key driving factors of phytoplankton communities in the Lake Qinghai, which can provide theoretical support for the subsequent protection and management of the Lake Qinghai.