Abstract: In order to clarify the microflora structure in Cheddar cheese processing, MiSeq high-throughput sequencing technology was used to analyze the community structure of Cheddar cheese at three stages of processing (post-pasteurization, curdling, and ripening 0, 30, 60 and 90 d) in this study. The results showed that the community structure varies widely of cheddar cheese during processing. The highest microbial community diversity and abundance were found after pasteurization (Chao1 index and Shannon index mean values were 6.09 and 1415.78, respectively). The dominant microflora in the pasteurization stage at the genus level was Stenotrophomonas (21.04%). The community structure was relatively similar in the curd and ripening stages, Lactococcus were the dominant flora in both stages, with abundance averaging more than 85%. During the ripening period, the relative abundance of Lactococcus increased first and then decreased. The community structure in the pasteurized cheeses was different compared to the other groups, and there was less change in the community structure of the groups during the ripening period. This study provides a basis for clarifying the community structure of Cheddar cheese, and has a certain reference value for the expansion of Cheddar cheese microbiome information.