The upper reaches of the Han River are the source region of water for the Middle Route of China's South-to-North Water Diversion Project, mainly for household, industrial, and irrigation purposes. Planktonic bacteria are more sensitive than macroorganisms to water physical and chemical properties and play a critical role in biogeochemical processes in river ecosystems. In November 2017 and April 2018, a systematic and methodical survey was carried out to evaluate the water quality and bacterial communities, on the mainstem of the Han River and its ve main tributaries. In this study, high-throughput sequencing technology has been employed to investigate the bacterioplankton community composition. The results indicated that: 1) Diversity increased downstream, especially in the upper reaches of the Han River. 2) The relative abundance of Actinobacteria increased with the increase of river length, while that of Bacteroidetes decreased slightly. 3) Five tributaries were found to be importance sources of taxa to the Han River, however in both months a large proportion of OTUs (37.84% and 36.34%, respectively) had unknown sources. 4) Finally, redundancy analysis (RDA) and Bioenv analysis showed that environmental parameters (pH, TN, Cond, NH 4 + -N, DO, NO 2 − -N, Chl-a and T) had a great in uence (p ≤ 0.05) on the bacterioplankton community. These research results are bene cial for the managing the ecological system, protecting the tributary biodiversity, and conserving the mainstem and tributaries of the Han River basin.