[Objective] In order to study the endophytic bacterial community structure and diversity of tobacco seeds.[Methods] The V3-V4 region of endophytic bacteria 16S rRNA gene of three varieties tobacco seeds was amplified, and the amplified fragments were sequenced using Illumina MiSeq high-throughput sequencing technology. The endophytic bacterial community structure and diversity of three varieties tobacco seeds were analyzed.[Results] We obtained a total of 128558 high-quality sequences in V3-V4 region from three varieties seeds, and the Shannon indexes varied in the range from 2.03 to 3.73. The endophytic bacterial community diversity indexes of K326 and Yunyan 85 were both higher than that of Yunyan 87. Proteobacteria, Actinobacteria, Firmicute and Bacteroidete were the dominant bacterial phylum of endophytic bacteria of three varieties tobacco seeds. The total number of endophytic bacteria genus in three varieties tobacco seeds was 27. Pseudomonas was the most dominant genera of endophytic bacteria of K326 and Yunyan 85, Escherichia-shigella was the most dominant genera of endophytic bacteria of Yunyan 87. 16S function prediction revealed that a large amount of beneficial function information about synthesis of proteins, nucleotides, sugars, coenzymes and metabolites showed higher abundance in tobacco seeds.[Conclusion] The diversity of endophytic bacteria of tobacco seeds was rich, the composition was basically similar, but their abundances showed some differences. Potential beneficial bacteria presented in seeds included Pseudomonas, Paenibacillus, Rhizobium, Massilia, Luteimonas, Salana and Lelliottia, which have a large number of beneficial metabolism-related functions. These research results could provide some reference information for the functional research and utilization of tobacco seed endophyte and biological control of seed diseases.