Intratumoral heterogeneity (ITH) is responsible for the majority of difficulties encountered in the treatment of lung-cancer patients. Therefore, the heterogeneity of NSCLC cell lines and primary lung adenocarcinoma was investigated by single-cell mass cytometry (CyTOF). First, we studied the single-cell heterogeneity of frequent NSCLC adenocarcinoma models, such as A549, H1975, and H1650. The intra- and inter-cell-line single-cell heterogeneity is represented in the expression patterns of 13 markers-namely GLUT1, MCT4, CA9, TMEM45A, CD66, CD274 (PD-L1), CD24, CD326 (EpCAM), pan-keratin, TRA-1-60, galectin-3, galectin-1, and EGFR. The qRT-PCR and CyTOF analyses revealed that a hypoxic microenvironment and altered metabolism may influence cell-line heterogeneity. Additionally, human primary lung adenocarcinoma and non-involved healthy lung tissue biopsies were homogenized to prepare a single-cell suspension for CyTOF analysis. The CyTOF showed the ITH of human primary lung adenocarcinoma for 14 markers; particularly, the higher expressions of GLUT1, MCT4, CA9, TMEM45A, and CD66 were associated with the lung-tumor tissue. Our single-cell results are the first to demonstrate TMEM45A expression in human lung adenocarcinoma, which was verified by immunohistochemistry.