The Chishui River is the last undammed tributary of the upper Yangtze River, extends cross Sichuan, Yunnan and Guizhou provinces, and it is the significant water source of Maotai liquor, Southwest of China. We investigated microbial community of the Chishui River in the Maotai town section, because of deep relationship between the water and the most famous Chinese liquor, Maotai liquor. In this study, diversities of bacteria and archaea of four seasons were analyzed in two different sampling sites using a barcoded 16S rRNA gene-pyrosequencing approach. The results show that the predominant community among all bacteria is Proteobacteria (70.16-94.29 %), of which Gamma-proteobacteria made up the largest portion. Bacterial community structure in spring and autumn tended to group together, and the operational taxonomic units of bacteria peaked in summer. The quantitative PCR (q-PCR) results revealed significantly higher number of gene copies in the downstream than that in the upstream, and were slightly higher in summer and spring than other seasons. Archaeal community structures had no obvious regular pattern, and species richness was higher in downstream in all seasons. Euryarchaeota and Thaumarchaeota were the dominant groups in archaeal populations, and abundant ammonia-oxidizing archaea was detected. The study significantly improved our understanding of microbial community in Maotai section of the Chishui River, where the unique and world-famous Maotai liquor is produced.