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Background: DNA methylation has been suggested as a biomarker for early cancer detection and treatment. Varieties of technologies for detecting DNA methylation have been developed, but they are not sufficiently sensitive for use in diagnostic devices. The aim of this study was to determine the suitability of Raman spectroscopy for label?free detection of methylated DNA. Methods: The methylated promoter regions of cancer?related genes cadherin 1 (CDH1) and retinoic acid receptor beta (RARB) served as target DNA sequences. Based on bisulfite conversion, oligonucleotides of methylated or nonmethylated probes and targets were synthesized for the DNA methylation assay. Principal component analysis with linear discriminant analysis (PCA?DA) was used to discriminate the hybridization between probes and targets (methylated probe and methylated target or nonmethylated probe and nonmethylated target) of CDH1 and RARB from nonhybridization between the probe and targets (methylated probe and nonmethylated target or nonmethylated probe and methylated target). Results: This study revealed that the CDH1 and RARB oligo sets and their hybridization data could be classified using PCA?DA. The classification results for CDH1 methylated probe + CDH1 methylated target versus CDH1 methylated probe + CDH1 unmethylated target showed sensitivity, specificity, and error rates of 92%, 100%, and 8%, respectively. The classification results for the RARB methylated probe + RARB methylated target versus RARB methylated probe + RARB unmethylated target showed sensitivity, specificity, and error rates of 92%, 93%, and 11%, respectively. Conclusions: Label?free detection of DNA methylation could be achieved using Raman spectroscopy with discriminant analysis.