Soil microbial communities play an important regulatory role in soil structural formation, nutrient cycling, and plant growth in terrestrial ecosystems, which are of great significance to the reestablishment of functions in degraded ecosystems. The dry-hot valley region of Jinsha River is an important ecological barrier in the upper reaches of the Yangtze River, and a typical ecologically fragile area. However, the understanding of variations in soil microbial community structure and driving factors in this region remains unclear. Here, the variations in soil microbial biomass and community structure of typical dry-hot valley across five sites along the Jinsha River basin were investigated using phospholipid fatty acid (PLFA) analysis. Further, the effects of climatic, vegetation and edaphic factors on soil microbial community structure were explored. The results showed that total soil microbial biomass ranged from 4.30 nmol/g to 13.20 nmol/g across the five sites. Bacterial biomass accounted for the largest proportion of total microbial biomass, with gram-positive bacterial (G+) and gram-negative bacterial (G-) biomass accounting for 26.22%-33.65% and 18.35%-23.67%, respectively. The proportion of fungal biomass in total microbial biomass was between 9.02% and 15.13%. These sites had significant difference in microbial biomass of total and specific functional group microorganisms, fungi/bacteria, G+/G-, cyclopropane fatty acids/monoenoic precursors, normal saturated fatty acids/monounsaturated fatty acids. Moreover, their soil microbial community structure formed different clusters based on the non-metric multidimensional scale (NMDS) analysis, suggesting significant differences among the sites. The results of variation partitioning analysis (VPA) revealed that the variation of soil microbial community structure was mainly driven by edaphic factors, followed by vegetation and climatic factors with the pure effects being 37%, 16% and 2%, respectively. Specifically, herb biomass (P=0.002), soil NO^-₃ (P=0.002), soil total carbon (P=0.003), pH (P=0.013) and soil water content (P=0.029) had significant effects on soil microbial community structure. Generally, herb biomass, soil total carbon and NO^-₃ content had positive effects, while soil water content and pH had negative effects on the PLFAs contents of total microbes and specific functional groups. In addition, the microbial PLFAs of various functional groups were also affected by the ratios of total carbon to total nitrogen and the ratio of total nitrogen to total phosphorus. The mean annual precipitation of climate factors had a negative impact on soil microbial biomass. Overall, the results revealed site-induced shifts in edaphic factors drove the variations in soil microbial community structure, and found the important role of herb biomass, which had vital implications for vegetation restoration in dry-hot valley region.