Background: Tumor-infiltrating immune cells analyzed by immunohistochemistry are reported as alternative prognostic factors to supplement TNM staging in operable non-small cell lung cancer (NSCLC), but little is done by flow cytometry. Method: We established a protocol to analyze immune cells in tumor, distant lung (far from tumor) and peripheral blood mononuclear cells (PBMCs) by 8 color flow cytometry. 30 NSCLC patients with adenocarcinoma, squamous cell carcinoma and more uncommon histotypes were included and statistical analyses of cell subsets were performed. Results: We have identified the following tumor-infiltrating immune cells: CD45+ leukocytes, granulocytes, CD19+ B cells, CD4+ and CD8+ T cells with naïve/memory phenotype, CD56+CD16+ and CD56+CD16- NK cells, CD14+ macrophages, CD123+ plasmacytoid dendritic cells (pDCs), CD1c+ myeloid dendritic cells (mDCs) and CD141+ mDCs. There was an increase in leukocyte numbers in tumor compared with distant lung in adenocarcinoma. When expressed as percent of total leukocytes, we found an increase in B cells in tumor compared with distant lung in both adenocarcinoma and squamous cell carcinoma. Squamous cell carcinoma tumors had a decrease of CD141+ mDCs compared with distant lung. Tumor-infiltrating immune cells between histotypes did not reveal differences in composition. TNM stages did not affect tumor-infiltrating immune cell composition in adenocarcinoma. Smoking history showed a decrease in CD141+ mDCs in present compared with previous smokers. PD-1 staining indicates strong variation between patients. Conclusions: Tumor microenvironment recruits immune cells and have a different composition than distant lung. Different composition of tumor-infiltrating immune cells between patients may be of future prognostic value.