Methylation of DNA is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells. The development of a simple and convenient method for site-specific discrimination of cytosine methylation is imperative for genomic studies. Here we report a facile method for distinguishing between cytosine and 5-methylcytosine. Osmium tetroxide caused the dihydroxylation of the C5-C6 double bond of 5-methylcytosine under an appropriate reaction conditions. The oxidation of 5-methylcytosine-containing target DNA was controlled by hybridization with a guide DNA. This technique facilitates the typing of cytosine methylation at a specific site of the target DNA.