Abstract: To investigate the diversity and distribution of microorganisms in reconstituted tobacco production process, 25 material samples were collected from a reconstituted tobacco production line, and the V3-V4 region of bacterial 16S rRNA and the fungal ITS region were sequenced by Illumina high-throughput sequencing technology. The results showed that: 1)A total of 393 bacterial genera and 165 fungal genera were identified, and among them the dominant genera were Lactobacillus, Bacillus, Candida and Aspergillus. 2) Significant differences were observed in the diversity and structure of bacterial and fungal communities among these material samples, and cluster analysis suggested that the differences of bacterial community diversity and structure among the bacterial genera were relatively small while those among the fungal genera were relatively large. 3)In the processes from extraction to coating solution preparation, the relative abundance of Lactobacillus increased and that of Candida decreased gradually.