In this study, we investigated the bacterial diversity and community structure of the Niyang River by high-throughput sequencing, and found that there were significant differences in the bacterial community structure among the upper, middle, and lower reaches of the Niyang River, as well as among the tributaries. The bacterial community of the Niyang River was mainly composed of the taxa of Proteobacteria, Actinobacteria, Bacteroidota, Cyanobacteria, and Planctomycetota. The dominant species were mostly unknown species, including Sporichthyaceae_sp. , Pseudarcicella-sp. , Limnohabitans_sp. , Flavobacterium_sp. , Candidatus_planktophila, Rhodoferax_sp. , Candidatus_nanopelagicus, f-Ilumatobacteraceae, and hgcI-sp. , the discovery of a large number of unknown species provides possibilities for the study of ancient bacterial diversity on the Qinghai Tibet plateau and the utilization of related biological functions. The number of ASVs in the upper Niyang River was significantly higher than that in other river segments, and α-diversity index analyses showed that its Shannon, ACE, Simpson, pielou, and Chao1 indices were significantly higher than those in other segments, suggesting that the upper Niyang River had the most abundant bacterial diversity in the water column. Neutral community model analyses showed that stochastic processes are more closely related to the bacterial community construction process in the Niyang River than deterministic processes, and that the differences in bacterial flora in different sections were mainly influenced by species substitution. Ecological niche analysis further disclosed that the Bahe River exhibited the highest mean niche breadth yet the lowest mean niche overlap value, suggesting a relatively low level of competition among the dominant bacterial species in this tributary. In conclusion, the Niyang River harbors abundant bacterial resources, which is of great significance for the study of bacterial diversity in the Qinghai-Tibet Plateau and the development and utilization of functional bacteria in the extreme environment of the plateau.