Abstract: Holopogon pekinensis X. Y. Mu & Bing Liu, a rare saprophytic orchid unique to North China, is distributed in mixed-wood forests at an altitude of about 1100 m with large habitat variety, which is significantly different from other saprophytic orchids (usually under birch forests above 1600 m) with large habitat differences. In this study, rhizosphere soil samples from two populations of H. pekinensis in the Beijing Yudu and Baihua mountains were collected to analyze microbial diversity using high-throughput sequencing. Based on the 4973 bacterial operational taxonomic units (OTUs) identified, the dominant bacterial groups consisted of seven phyla, including Proteobacteria, Actinobacteria, and Bacteroidetes, and the dominant genera included MND1, Nitrospira, and Haliangium. Based on the 1914 fungal OTUs identified, the dominant fungal groups were Ascomycota, Basidiomycota, and Zygomycota and the dominant genera were Archaeorhizomyces, Sebacina, and Mortierella. Most of them are ectomycorrhizal fungi and consistent with the existing reports of orchid mycorrhizal fungi, so they are most likely to be the potential mycorrhizal fungi of H. pekinensis. Diversity index analysis indicated that fungal and bacterial community richness and evenness in the rhizosphere soil were higher in the Yudu Mountain population than in the Baihua Mountain population. Soil microbial diversity showed a certain correlation with arbor species diversity in the two H. pekinensis populations.