Abstract: The representative tobacco fields covering 88 tobacco-growing counties(districts) of 12 prefecture-level cities were selected as research objects. A total of 564 topsoil samples were collected, and nine soil nutrient indicators were determined, including pH, soil organic matter, alkali-hydrolysable nitrogen, water-soluble chlorine, total nitrogen, available phosphorus, available potassium, total phosphorus and total potassium. Multiple comparisons were performed to analyze the differences in each soil nutrient indicator among tobacco-growing regions. Hierarchical cluster analysis was adopted to clarify the spatial differentiation grouping characteristics of soil nutrients in different prefecture-level cities. Furthermore, fuzzy mathematics membership functions combined with principal component analysis(PCA) were used to quantify the integrated fertility index(IFI) of soil. The results showed that:(1) Significant regional variations existed in soil nutrients of tobacco-growing areas in Yunnan, with coefficients of variation(CV) of the nine indicators ranging from 12.17% to 81.92%. Total phosphorus, available phosphorus and water-soluble chlorine exhibited extremely uneven spatial distributions. The pH value, organic matter, total nitrogen, available phosphorus and available potassium fell within the optimal ranges. Alkali-hydrolysable nitrogen was generally excessive, while total phosphorus, total potassium and water-soluble chlorine were insufficient in most regions.(2) Cluster analysis showed that the spatial distribution of soil nutrients failed to follow geographical adjacency, indicating that micro-environmental conditions and anthropogenic management exerted stronger impacts on soil properties than geographical proximity.(3) Soils with medium-high and medium fertility dominated all tobacco-growing areas, accounting for 53.72% and 36.17% of the total samples, respectively. High-fertility and low-medium-fertility soils accounted for 5.32% and 4.79%, respectively. Chuxiong and Lijiang presented relatively low integrated soil fertility. In conclusion, it is recommended to increase the application of phosphorus and potassium fertilizers and strictly control nitrogen input in low-fertility zones, while maintaining the current fertilization regimes in high-fertility zones, so as to support the green and high-quality development of the flue-cured tobacco industry.