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Rushan is a traditional dairy product consumed by the Bai people in the Yunnan Province of China, and its production still follows the traditional procedure of backslopping. However, how the microbial composition of raw materials and processing shape the microorganisms in rushan have not been systemically reported. In this study, high-throughput sequencing technique was applied to analyze the microbial compositions of raw milk, fresh rushan, curd whey, acid whey, and dry rushan at the phylum, family, genus, and Lactobacillus species levels. The results indicated that Lactobacillus , Lactococcus , and Streptococcus were dominant genera in rushan, whereas Lactobacillus kefiranofaciens and Lactobacillus helveticus were the 2 abundant species at the Lactobacillus species level. The network analysis indicated that raw milk mainly contributed to the microbial diversity of rushan, whereas acid whey made a great contribution to shaping the relative abundance of microbes in rushan and dramatically increased acid-producing genera, such as Lactobacillus and Acetobacter. The variation in microbial composition led to an increase in the relative abundance of pathways related to energy supply, acid production, fatty acid accumulation, cysteine, methionine, and lysine accumulation. The volatile profile of rushan was rich in esters and acids, and the high relative abundance of Lactobacillus might be associated with reduction of amino acid metabolism, degradation of unpleasant flavored xylene, and accumulation of decanoic, dodecanoic, and tetradecanoic acids in the products. The accumulation of medium long-chain fatty acids might result from the relative abundance of FabF , FabZ , and FabI , particularly from Lactobacillus amylolyticus and Lacticaseibacillus paracasei. [Display omitted] [ABSTRACT FROM AUTHOR]