Background: Lung cancer is the leading cause of cancer-related death worldwide. Short stature homeobox 2 (SHOX2) methylation detected by real-time polymerase chain reaction (PCR) has recently been demonstrated to be a potential biomarker in the diagnosis of lung cancer. However, more cost-effective methods are still needed to help cancer detection in the early stage of lung cancer. The aim of this study was to examine the methylation status of the SHOX2 gene and to investigate its diagnostic value in non-small cell lung cancer (NSCLC) patients. Methods: A total of 89 Chinese NSCLC patients and 9 non-tumor patients was enrolled in this study. The methylation status of SHOX2 gene in NSCLC tumor tissues/corresponding non-neoplastic lung tissues and lung tissues from non-tumor patients was examined by methylation-specific PCR (MSP). Results: We found that SHOX2 methylation was significantly associated with NSCLC (P=0.003). We also analyzed the correlation of SHOX2 methylation with clinicopathological variables including sex, age, tumor pathologic classification, tumor differentiation degree, TNM stage, T stage, and nodal status, and found no significant correlation between them. Conclusions: These results suggested that SHOX2 gene methylation was closely associated with lung carcinogenesis. Thus, SHOX2 methylation could be used as a potential marker to help NSCLC detection. MSP might be used as a cost-effective method alternative to real-time PCR in detection of SHOX2 methylation in the early diagnosis of NSCLC.