Abstract:[Objective] This study aims to analyze the structure and influencing factors of bacterial community in the rhizosphere soil of highland barley in different planting areas in Xizang, reveal the biological markers of rhizosphere bacteria in specific environments, and provide a reference for the exploration and research of rhizosphere growth-promoting bacteria and their roles. [Methods] We carried out 16S rRNA gene high-throughput sequencing and statistical data analysis to compare the composition and structures of the bacterial communities in the rhizosphere soils of highland barley cultivated in five cities of Xizang. We then analyzed the biological markers and the driving factors of community structure changes in the rhizosphere bacteria of highland barley. [Results] The sequencing of 45 rhizosphere soil samples yielded 10 715 operational taxonomic units (OTUs), which belonged to 2 783 species, 1 244 genera of 43 phyla. Actinobacteriota, Proteobacteria, Chloroflexi, Acidobacteriota,Bacteroidota, Firmicutes, Gemmatimonadota, Myxococcota, and Patescibacteria were the dominant bacterial phyla, with the relative abundance of 94.92%-96.56%. The community structure of rhizosphere bacteria showed significant differences among the five cities, with greater inter-group differences than intra-group differences (R=0.226 9, P=0.001). Actinobacteriota, Chloroflexi, Acidobacteriota, Bacteroidota, and Patescibacteria showed different relative abundance among the five cities (P<0.05). We identified potential biological markers in the rhizosphere soil of barley in all five cities. There were potential biological markers in the rhizosphere soil of highland barley in the five cities. Lasa and Shannan had only three and six unique bacterial clades, respectively, with more complex co-occurrence network and tighter connections between OTUs. The key phyla in the rhizosphere of highland barley were Proteobacteria, Chloroflexi,Actinobacteriota, and Acidobacteriota. Additionally, Endobacteria,Methylomirabilota, and Cyanobacteria were unique taxa in Linzhi, Rikaze, and Shannan, respectively. The changes in the bacterial community structure in the rhizosphere of highland barley were mainly related to environmental factors such as pH, total potassium, available potassium, carbon to phosphorus ratio, and altitude, with total potassium being the most important factor (r2=0.621 4, P=0.001). [Conclusion] The bacteria in rhizosphere soil of highland barley in Xizang have high diversity, with significant differences among the five cities. Additionally, different growing areas of highland barley have unique biological markers of bacteria in the rhizosphere. The findings provide a reference for revealing the role of unique rhizosphere bacteria in the growth and environmental adaptation of highland barley and for exploring excellent rhizosphere plant-promoting bacteria.