[Background] The traditional koumiss is naturally fermented by fresh mare’s milk, and the quality of koumiss is greatly affected by the bacteria in fresh mare’s milk. However, the microbiota composition in fresh mare’s milk was rarely investigated. [Objective] In order to explore the microbial variation from fresh mare’s milk to koumiss, microbial diversity of fresh mare’s milk was investigated, to provide a reference for the industrialized production of koumiss. [Methods] Six fresh mare’s milk samples were collected from herdsmen families of Xilinhot in Inner Mongolia, China. The bacterial full-length 16S rRNA gene sequences in all samples were sequenced by PacBio SMRT sequencing technology to analyze the bacterial composition and diversity. Meanwhile, the structure and diversity of bacteria in fresh mare’s milk and koumiss were compared. [Results] The results showed a large number of lactic acid bacteria was detected in the fresh mare’s milk such as Lactococcus lactis (12.43%), Lactococcus garvieae (3.53%) and Leuconostoc mesenteroides (1.77%). In addition, some opportunistic pathogens were also detected such as Cronobacter sakazakii. Compared with koumiss, the bacterial diversity in fresh mare’s milk were significantly higher, and Lactococcus lactis and Lactobacillus helveticus were found both in fresh mare’s milk and koumiss. In fresh mare’s milk, Leuconostoc mesenteroides correlated with Lactococcus lactis and Lactococcus garvieae positive, negative related with Aeromonas hydrophila, which implied lactic acid bacteria may suppress pathogen bacteria during the fermentation. [Conclusion] During the fermentation of koumiss, the growth of lactic acid bacteria could inhibit pathogen bacteria, decrease the bacterial diversity and improve the quality of koumiss.