Abstract: In this experiment, the natural starter of koumiss in Xilin Gol pastoral area was used as the research object to explore its flavor substances and microbial diversity. Liquid - liquid extraction - gas chromatography-mass spectrometry (LLE-GC-MS) was used to determine the flavor compounds and Illumina MiSeq high-throughput sequencing technology was used to sequence and analyze the V3-V4 variable region of bacterial 16S rRNA gene and ITS region of fungal gene, and their related metabolic pathways were analyzed and predicted. The results showed that a total of 43 flavor substances were detected in the natural starter, the main flavor substances were L-lactic acid, palmitic acid, lauric acid, ethyl caprylate, ethyl lactate, ethyl laurate, 2, 3-butanediol, dodecanethiol, isoamyl alcohol, etc. A total of 180069 bacterial and 187397 fungal sequences were obtained by high-throughput sequencing. The bacterial community mainly included Lactobacillus, Acetobacter, Lactococcus and Streptococcus, among which Lactobacillus was the dominant bacterium and the relative abundance was 72.26%. The fungal community mainly consisted of Dekkera and Kluyveromyces, among which Dekkera was the dominant fungal genus with a relative abundance of 94.09%. According to the prediction of gene function, carbohydrate metabolism and amino acid metabolism of starter were prominent, and there were a lot of enzyme genes in the metabolic pathway, which would lay a genetic foundation for the formation of flavor substances in the fermentation process of koumiss.