[Objective] To study the diversity of endophytic diazotrophic bacteria in flue-cured tobacco, explore the resource of endophytic diazotrophic bacteria, and enrich the gene pool of endophytic diazotrophic bacteria. [Methods] Pure culture, transcription factor amplification (BOX-PCR), 16S rRNA gene sequencing, and phylogeny analysis were applied to study the diversity and phylogeny of endophytic diazotrophic bacteria strains isolated from flue-cured tobacco leaf. The nitrogenase activities, phosphorus and potassium solubilizing activities and indole-3-acetic acid (IAA) production of the isolates were also evaluated. [Results] totally, 62 endophytic diazotrophic strains were isolated by using Ashby culture media. Based on the BOX-PCR patterns, 16 typical strains were selected for further 16S rRNA gene sequencing. Phylogeny analysis results based on the 16S rRNA gene sequences showed that the isolates were affiliated to 3 genera, including Bacillus, Pantoea, and Curtobacterium. Bacillus was the dominant species among all the isolates. 32.3% (20/62) of the isolates exhibited nitrogenase activities, 12.9% (8/62) showed abilities in producing IAA, 6.5% (4/62) had phosphate solubilizing activities and 4.8% (3/62) possessed potassium releasing activities. [Conclusion] Abundant culturable endophytic diazotrophic bacteria existed in flue-cured tobacco and showed a potential application prospect.