Soil microbial characteristics can sensitively reflect soil quality and health index. In order to illustrate the difference in microbial information between different ecological regions, tobacco-planted soils were collected from 31 regions in Guizhou Province and the microbial community structure of soil was assessed and analysed by high throughput sequencing (16S V4 region). The Operational Taxonomic Units (OTUs) were significantly diverse in different ecological regions. The soil microbial population in the east was relatively higher than that in the west, while the north was higher than the south. Besides, the number of OTUs in north Guizhou Province was the highest, while that in northwest Guizhou Province was the lowest. The results showed that Proteobacteria and Actinobacteria were the top two microbial phyla in relative abundance in north Guizhou Province. In the middle area of Guizhou Province, Proteobacteria abundance was significantly denser than Actinobacteria abundance. The soil dominant genera in the southwest and middle district were much more than other regions in Guizhou Province. However, the microbial population was unbalanced in this area, raising the potential for soil-borne disease outbreaks. Furthermore, Streptomyces, Thiobacillus and Pseudomonadaceae accounted for a great proportion in the middle and the east region, the southeast area and Tianzhu district, respectively. Soil microbial community information will be better understood through this investigation and analysis. These results will supply a theoretical basis and build a good foundation for soil bio-remediation and sustainability in tobacco leaf production afterwards.