Objective To investigate the species and microbial diversity of ticks from three autonomous prefectures in Guizhou Province. Methods In April 2019 and July 2020, parasitic ticks on the body surfaces of cattle, sheep and rodents were collected in Qiandongnan Miao and Dong Autonomous Prefecture (referred to as Qiandongnan), Qiannan Buyi and Miao Autonomous Prefecture (referred to as Qiannan) and Qianxinan Buyi and Miao Autonomous Prefecture (referred to as Qianxinan) in Guizhou Province, followed by morphological identification. The ticks were divided into 6 groups based on the collection regions and tick species. After DNA extraction, 16S rDNA high-throughput sequencing was performed. The sequencing results were analyzed by operational taxonomic unit (OTU) classification cluster and compared with the ribosomal database to obtain species annotations, enabling bacterial community composition analysis and α diversity analysis. Hierarchical clustering was conducted based on the β diversity distance matrix and a sample clustering tree was constructed using the Bray-Curtis algorithm. Non-metric multidimensional scale (NMDS) analysis and inter-group similarity analysis were performed using R 3.6.3 software. Results A total of 1 463 ticks were collected, including 1 227 Rhipicephalus microplus (83.87%), 208 Haemaphysalis longicornis (14.22%), 25 Ixodes granulatus (1.71%) and 3 Haemaphysalis flava (0.20%). In Qiandongnan, Qiannan and Qianxinan, 220, 1 039 and 204 ticks were collected respectively, accounting for 15.04%, 71.02% and 13.94%. A total of 1 682 OTUs were generated. α diversity analysis revealed that H. longicornis from Qiandongnan exhibited relatively high Shannon index, Chao1 index, Ace index and evenness index values of 4.868, 568.481, 567.479 and 0.770, respectively. Bacterial community identification annotated a total of 29 phyla, 70 classes, 118 orders, 245 families and 503 genera. Proteobacteria, Alphaproteobacteria, Rickettsiales, Rickettsiaceae and Rickettsia emerged as the dominant taxa at their respective taxonomic levels, with average relative abundances of 75.52%, 57.70%, 52.47%, 50.88% and 50.88%, respectively. The bacterial genera carried by R. microplus were predominantly Rickettsia (89.45%) and Coxiella (2.82%), while H. longicornis mainly harbored Rickettsia (18.06%) and Pseudomonas (11.98%), and I. granulatus primarily carried Spiroplasma (33.19%) and Staphylococcus (22.22%). The dominant bacterial genus in ticks from Qiandongnan, Qiannan, and Qianxinan was Rickettsia, with average relative abundances of 46.96%, 60.20% and 45.47%, respectively. The sample clustering tree demonstrated that R. microplus samples from 3 regions clustered together with a H. longicornis sample from Qiannan, while the remaining H. longicornis samples formed a separate cluster, and 3 I. granulatus samples from Qianxinan clustered independently. NMDS and inter-group similarity analysis indicated distinct bacterial community compositions among the 6 sample groups (R = 0.599, P < 0.01). Conclusion The bacterial communities carried by ticks in the three autonomous prefectures of Guizhou Province exhibited rich diversity, with Rickettsia being the dominant genus. Additionally, the bacterial community compositions differed among various tick species.