Abstract: To investigate the variation of microorganisms in aqueous extract of waste tobacco scrap during paper-making process of reconstituted tobacco sheet materials, Roche 454 FLX pyrosequencing was adopted to analyze the diversity of 16S rRNA gene sequences of bacteria in the aqueous extract of waste tobacco scrap. The variation of bacterial communities and their abundances in the extracts was obtained. The results showed that:1) The aqueous extracts of waste tobacco scrap, including the original extract (M1), the concentrated extract (M2) and the aged extract (M3), harbored abundant levels of bacteria. The dominant phyla was Firmicutes. Lactobacillus and Lysinibacillus were the dominant genera of Firmicutes in all the samples. 2)It was also found that the abundance of Lactobacillus decreased from M1 to M3, while the abundance of Lysinibacillus increased. The variation of bacterial community based on high-throughout sequencing provides a scientific basis for improving the quality of paper-making reconstituted tobacco materials.