Abstract: In order to study the community structure of bacterioplankton and the driving factors in the mainstream of Yangtze River, Quantitative Real-time PCR and high-throughtput sequencing targeting on the bacterial 16S rRNA gene were used. Water samples were collected from 40 sites from Luzhou (Sichuan province) to Wusongkou (Shanghai city) along the mainstream of Yangtze River. The results show that the 16S rRNA gene abundance of all sites range from 2.64×106 copies/L to 1.44×109 copies/L. There is no significant difference in α-diversity between upper (from Luzhou to Zigui), middle (from Yichang to Hukou) and lower (from Huayang to estuary) reaches. Meanwhile, the dominant phyla in the mainstream of Yangtze River are Proteobacteria (7.49%~86.53%), Actinobacteria (0.27%~54.72%), Firmicutes (0.03%~90.95%), Bacteroidetes (0.50%~45.36%) and Deinococcus-Thermus (0.00%~23.96%). The dominant taxa at genus or family level include Comamonadaceae (0.03%~65.67%), CL500-29_marine_group (0.00%~24.23%), hgcI_clade (0.00%~28.82%)、Bacillus (0.00%~90.88%) and Hydrogenophaga (0.03%~38.55%). Moreover, the taxa with the highest relative abundance in upper reaches is Bacillus (0.00~90.88%), while it is Comamonadaceae in middle and lower reaches (0.60%~65.67% and 0.03%~65.67%, respectively). Additionally, the driving factors of these differences of bacterioplankton community structure vary in three reaches. The key factors affected the community structure are pH and suspend solids (SS) in upper reaches, dissolved oxygen (DO) in middle reaches, and DO, water temperature (WT) and total phosphorus (TP) in lower reaches.