Objective: Ticks are obligate blood-sucking vectors for multiple zoonotic diseases. In this study, tick samples were collected from Yunnan Province, China, which is well-known as the “Global Biodiversity Hotspot” in the world. This study aimed to clarify the microbial populations, including pathogens, associated with ticks and to identify the diversity of tick-borne microbiota in this region. Methods: The 16S rRNA full-length sequencing from pooled tick DNA samples and PCR amplification of pathogenic genera from individual samples were performed to understand tick-associated microbiota in this region. Results: A total of 191 adult ticks of 5 tick species were included and revealed 11 phyla and 126 genera bacteria, including pathogenic Anaplasma, Ehrlichia, Candidatus Neoehrlichia, Rickettsia, Borrelia, and Babesia. Further identification suggested that Rickettsia sp. YN01 was a variant strain of Rickettsia spp. IG-1, but Rickettsia sp. YN02 and Rickettsia sp. YN03, were potentially two new SFGR species. Conclusions: This study revealed the complexity of ecological interactions between host and microbe and provided insight for the biological control of ticks. A high microbial diversity in ticks from Yunnan was identified, and more investigation should be undertaken to elucidate the pathogenicity in the area.