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Simple Summary: The gut microbiota are crucial for hosts. For mammals, different gastrointestinal tract (GIT) segments have specific microbial communities, which play an essential role in the host's nutrition, metabolism, immunity, and health. Plateau zokors (Eospalax baileyi) and Gansu zokors (Eospalax cansus) are closely related species that belong to the Spalacidae family, and are common pests in agriculture, forestry, and animal husbandry in northwestern China, with a sympatric distribution area in the transition zone between the Qinghai-Tibetan Plateau and the Loess Plateau. Here, the characteristics of the microbiota communities in different GIT segments of the plateau zokor and the Gansu zokor were studied, and the microbiota communities of the two zokor species were compared. Our results provide important information for further study on the function of microbiota communities in different GIT segments and the potential use of the gut microbiota as a new method for the population management of the zokors. In this study, based on high-throughput sequencing technology, the biodiversity and the community structure of microbiota in different GIT segments (the stomach, small intestine, cecum and rectum) of plateau zokors and Gansu zokors were studied and compared. A source tracking analysis for the microbial communities of different GIT segments was carried out using the fast expectation–maximization microbial source tracking (FEAST) method. We found that, for both species, the microbial community richness and diversity of the small intestine were almost the lowest while those of the cecum were the highest among the four segments of the GIT. Beta diversity analyses revealed that the bacterial community structures of different GIT segments were significantly different. As for the comparison between species, the bacterial community compositions of the whole GIT, as well as for each segment, were all significantly different. Source tracking conducted on both zokors indicated that the soil has little effect on the bacterial community of the GIT. A fairly high percentage of rectum source for the bacterial community of the stomach indicated that both zokors may engage in coprophagy. [ABSTRACT FROM AUTHOR]