[Background] Stellera chamaejasme L. is one of the landmark plants of natural grassland degradation in China. The root endophytic microorganisms play an important role in the process of invasion. It is of great significance to explore the root endophytic microbial community to the utilization beneficial microbial resources, and to prevention and control of the Stellera chamaejasme L. [Objective] The purpose of this study was to explore the endophytic fungi and bacteria composition and diversity in the roots of Stellera chamaejasme L., a poisonous weed in different alpine grasslands at the eastern foot of Qilian Mountains in Gansu province. [Methods] The composition and diversity of endophytic fungi and bacteria in the roots of Stellera chamaejasme L. in different alpine grasslands at the eastern foot of Qilian Mountains in Gansu province were analyzed by Illumina MiSeq high-throughput sequencing technique. [Results] The results showed that Ascomycota in different alpine steppe was dominant in fungi in the roots of Stellera chamaejasme L. from different alpine grasslands. Cyanobacteria, Proteobacteria and Actinobacteria belong to the dominant groups in root endophytic bacteria. The operational taxonomic unit (OTU) number and Shannon diversity index of fungi and bacteria in G4 sampling site were higher than those in the other four sampling sites. Principal coordinate analysis (PCoA) showed that there were some differences among endophytic fungi and bacteria communities in the roots of Stellera chamaejasme L. in different alpine grasslands. One-Way ANOVA analysis and Pearson correlation analysis showed that the community structure of Stellera chamaejasme L. fungi had little correlation at the genus level, while the bacteria had a certain correlation at the genus level.[Conclusion] The purpose of this study is to further develop and utilize the endophytic fungi and bacterial resources and provide theoretical reference for elucidating the community structure and ecological function of endophytic flora of Stellera chamaejasme L.