Abstract: This study investigated the diversity and community structure of endophytic bacteria in different parts (root, stem, and leaf) of the Qinyan96 tobacco cultivar. The V5-V6 region of the bacterial 16S rRNA gene was amplified and sequenced using Illumina MiSeq high-throughput sequencing technology to analyze differences in the endophytic bacterial communities among root, stem, and leaf tissues. A total of 2,123 operational taxonomic units (OTUs) were detected across all samples, belonging to 18 phyla, 40 classes, 103 orders, 168 families, and 323 genera. The number of core OTUs in roots was significantly higher than that in stems and leaves. Alpha diversity indices decreased in the order: root > stem > leaf. Beta diversity and clustering analyses indicated that the endophytic bacterial community structures of stems and leaves were similar to each other but distinct from those of roots. At the phylum level, Proteobacteria was the dominant phylum in all three tissues. LEfSe analysis revealed that the root tissues were significantly enriched with abundant genera, including Pseudomonas, Enterobacter, Ensifer, Chitinophaga, Delftia, and Variovorax. The stem tissues were significantly enriched with Burkholderia–Caballeronia–Paraburkholderia, Halomonas, Ralstonia, and Paucibacter. No significantly enriched bacterial genera were detected in leaves. PICRUSt2-based functional prediction indicated that endophytic bacteria in Qinyan 96 tobacco plants were predominantly involved in metabolic functions, with functional profiles mainly distributed across 30 categories of metabolic pathways in roots, stems, and leaves, suggesting potential application value in immune defense and environmental adaptation. This study reveals the diversity, community structure, and potential functions of endophytic bacteria in the Qinyan96 tobacco plants, providing a theoretical basis for the exploration and targeted application of beneficial endophytic bacteria.