The purpose of this study is to identify the characteristics of microbial communities in the lung cancer tissues from patients in Kunming sity of southwestern China and to compare the microbial differences at different clinical stages of lung cancer to uncover potential microbial biomarkers. In total, 40 tissue samples of primary lung adenocarcinoma were collected and further performed by 16S rRNA gene sequencing. The subjects were grouped according to TNM stages (T and N group), clinical stage, and smoke status, and the microbial differences in each group were compared. Analysis of sequence data to determine beta diversity, the UniFrac distance was calculated by QIIME and visualized by principal coordinate analysis (PCoA) using R (version 2.15.3). Microbiome abundance and diversity between different groups were calculated by t test or Wilcoxon rank sum test and drawn by R. The linear discriminant analysis effect size (LEfSe) method was utilized to compare relative abundances of all bacterial taxa between groups. A total of 951 OTUs were identified in the cancer tissues. No significant difference has been found in the alpha diversity within all the groups. Beta diversity significantly differed in the N, T, and clinical stage groups. By LEfSe analysis, eight differential taxa including Bifidobacterium were identified in the N group. In the T1 and T2 group, the LEfSe result identified five phyla and ten genera. The differential genera were Moraxella, Dolosigranulum, unidentified_Corynebacteriaceae, and Citrobacter in the T2 group and Bifidobacterium, Alistipes, Akkermansia, Blautia, Lactobacillus, as well as Faecalibaculum in the T1 group. Differential bacterial composition and abundance were also observed in the clinical stage group. This study confirmed that by 16S rRNA sequencing, we identified the dominant microbe of lung cancer tissue in different groups. Bifidobacterium may play an essential role in lymph node metastasis and tumor progression, providing a specific potential microbial biomarker for lung adenocarcinoma. PCR products were subject to vertical electrophoresis on 2% agarose gels, and a colloid recovery kit (Qiagen, Valencia, CA) was applied to recover the target bands. Libraries were generated by the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, USA), and the concentrations were quantitated with a Qubit fluorometer. Finally, the qualified libraries were sequenced by NovaSeq6000 (Illumina).© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.