Objective To evaluate the methylation levels of DNA at six specific CpG sites located in the mRNA region of histone cluster 4 subfamily F member 6 （HIST1H4F） gene and determine their diagnostic significance about lung cancer. Methods The DNA methylation levels of 15 cases of lung cancer and adjacent paired normal lung tissue were detected using pyrophosphate sequencing. Based on preliminary evaluation， a methylation-sensitive restriction enzyme-fluorescence quantitative PCR （MSRE-qPCR） method was developed to detect DNA methylation levels in the test group （60 cases of lung adenocarcinoma， 38 cases of squamous cell carcinoma， 30 cases of benign diseases， and 26 cases of normal lung tissue） and the validation group （36 cases of lung adenocarcinoma， 16 cases of squamous cell carcinoma， 21 cases of benign diseases， and 23 cases of normal lung tissue）. The diagnostic value was evaluated using ROC curves. Results The results of pyrophosphate sequencing showed that the methylation levels of lung cancer were significantly higher than that of paired normal lung tissue （P < 0.005）. The detection results of MSRE-qPCR showed that the areas under the ROC curve for diagnosing lung cancer in the test group and validation group were 0.894 and 0.888， with sensitivity of 76.5% and 73.1%， and specificity of 92.9% and 97.7%， respectively. The methylation levels were significantly positively correlated with smoking in lung cancer patients （r = 0.273， P < 0.01）. Conclusion The six CpG sites in the mRNA region of the HIST1H4F gene can serve as biomarkers for diagnosing lung cancer， providinga new molecular target for clinical lung cancer diagnosis.