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Background: DNA methylation on cytosine in the CpG dinucleotides is one of the most common epigenetic perturbations taking place during cancer initiation, progression, occurrence and resistance therapy. DNA methylation seems to be sufficiently stable epigenetic modification to be utilized as a cancer biomarker in in vitro diagnostic (IVD) settings. Nowadays, the SHOX2 methylation (mSHOX2) is one of the most valuable DNA methylation biomarkers of lung cancer that is the leading cause of cancer death. It is being continuously validated across ethnicities, lifestyles and lifespan. This study focused on characteristics of mSHOX2 in Vietnamese patients with lung cancer since a lack of investigation and evidence of its utility in this country. Methods: The probe and primer sets were designed according to the MethyLight method for quantitative assessment of the mSHOX2 in 214 formalin-fixed paraffin-embedded (FFPE) lung tissues and 57 plasma samples. Results: mSHOX2 in FFPE tissues allowed discriminating benign and malignant lung diseases with 60% (95% CI 50.7–68.8%) sensitivity and 90.4% (95% CI 82.6–95.5%) specificity. Importantly, based on mSHOX2 in plasma, lung cancer could be detected with 83.3% (95% CI 65.3–94.4%) sensitivity and 92.6% (95% CI 75.7–99.1%) specificity, respectively. There were insignificant associations between mSHOX2 with age, cancer stage, EGFR mutation and serum CEA, CYFRA21-1 concentrations except for that gender. Conclusion: Our study indicated that mSHOX2 was satisfactory for distinguishing malignant from benign lung tissue and noninvasively detecting lung cancer. [ABSTRACT FROM AUTHOR]