To investigate the relationship between the epigenetic alterations of genes encoding the key enzymes of homocysteine (Hcy) metabolism and the risk for atherothrombotic stroke (ATS) in the Chinese Han population. Methods: This case-controlled study enrolled 30 cases with ATS and 30 healthy controls. The CpG islands region of the targeted gene (5kb upstream region to exon 2) was identified. Detection of DNA methylation was accomplished by sequencing bisulfite-altered DNA. Univariate and multivariate logistic regression analysis were used to detect the differences in DNA methylation levels between the two groups. Results: DNA methylation analysis of the cystathionine-β-synthase (CBS) gene was abandoned because of the high homology of the 4 CpG island sequences. There were respectively 2 and 1 CpG islands found in the 5, 10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) genes. However, no change in DNA methylation levels was detected in the CpG islands of these two genes. Conclusion: Our study suggested no significant association between DNA methylation alterations of the MTHFR and MTR genes with ATS among the Han Chinese.