The Yarlung Tsangpo River is one of the world"s major international rivers and also one of the most sensitive and fragile ecosystems globally. Eukaryotic phytoplankton play a vital role in maintaining the ecological balance of this river ecosystem. To reveal the diversity, spatiotemporal distribution characteristics, and key driving factors of eukaryotic phytoplankton communities in the Nyang River and Palong Tsangpo, representative primary tributaries of the lower Yarlung Tsangpo River, this study employed high-throughput amplicon sequencing technology to investigate the diversity patterns and driving factors of eukaryotic phytoplankton across three seasons spring, summer, and autumn—in this watershed. Results revealed that 6,723 ASVs of eukaryotic phytoplankton were identified across the three seasons, belonging to 8 phyla, 39 classes, 87 orders, 100 families, and 265 genera. The Phaeophyceae phylum exhibited the highest abundance in species composition throughout all seasons. Overall, Shannon diversity, Simpson diversity, and Pielou"s evenness index exhibited a pattern of spring > summer > autumn, with highly significant seasonal differences (P < 0.001). Eukaryotic phytoplankton communities showed highly significant seasonal differences (P < 0.001). Summer eukaryotic phytoplankton community assembly was dominated by stochastic processes, while spring and autumn communities were dominated by deterministic processes. All three seasons exhibited highly significant geographic decay trends (P < 0.001), elevation decay trends (P < 0.001), and environmental decay trends (P < 0.001). Interactions among eukaryotic phytoplankton communities were predominantly cooperative. Environmental factors significantly explained more community variation than geographic and altitude factors across all seasons. Key environmental drivers for spring eukaryotic phytoplankton communities were pH, dissolved oxygen (DO), water temperature (WT), turbidity (TUR), ammonium nitrogen (NH??-N), and altitude (ALT); summer: electrical conductivity (EC) and water temperature (WT); autumn: water temperature (WT), dissolved oxygen (DO), and electrical conductivity (EC). Water temperature exerted a highly significant influence on eukaryotic phytoplankton communities across all three seasons. This study employed high-throughput amplicon sequencing to analyze the spatiotemporal structure of eukaryotic phytoplankton communities in the river basin. It revealed the distribution patterns and variation characteristics of eukaryotic phytoplankton communities in plateau rivers, providing crucial evidence for microbial diversity conservation and aquatic ecosystem health management in plateau regions.