Abstract The gut microbiome plays a crucial role in the development of multiple chronic diseases. Several solid tumors including lung cancer are also implicated in the gut microbiome in cancer initiation and progression, possibly affecting signaling pathways in the tumor microenvironment. However, the microbiome in the peripheral lung tissue is unexplored and their relationship to the lung tumor microenvironment is still unknown. Thus, to explore the relationship between the microbiome and the tumor microenvironment of lung cancer, we performed shotgun metagenomic sequencing of the lung cancer tissue, especially in lung cancer patients accompanied by chronic obstructive pulmonary disease (COPD). We confirmed the presence of a diverse microbiome in peripheral lung cancer tissue. Next, we focused on individual species. We observed significant differences in the relative abundance of several species between COPD and non-COPD patients, and cancer and non-cancer tissue. Then, we focused on the metagenomic diversity between samples. The number of species in COPD patients and cancer tissue were decreased. And the bacterial total abundance in each sample were observed similar trend. On the other hand, alpha diversity in each sample were showed different trend to them. In the analysis of beta diversity using Principal Coordinate Analysis (PCoA), clusters were formed in each group. These findings suggest that microbiome imbalance known as dysbiosis in the peripheral lung tissue may affect the tumor microenvironment resulting in cancer progression. Citation Format: Atsushi Matsuoka, Kazuhiko Shien, Shuta Tomida, Daisuke Mizuno, Masayoshi Ohki, Ryo Yoshichika, Tomohiro Higashihara, Naohiro Hayashi, Fumiaki Mukohara, Mao Yoshikawa, Ken Suzawa, Hiromasa Yamamoto, Shinichi Toyooka. Shotgun metagenomics of COPD-associated lung cancer tissue microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2800.