5-Methylcytosine (5mC) can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. Traditional bisulfite-based DNA methylation analysis techniques have been widely used in the detection of 5mC. However, they can not discriminate 5hmC from 5mC, leading to overestimate 5mC levels. We here introduce a strategy, combination of selective oxidation and bisulfite pyrosequencing (BS-Pyroseq), for quantification of both 5mC and 5hmC at CpG sites within the promoters of CDH1, DAPK, RARβ and RUNX3 genes in a panel of cell lines and clinical samples. As expected, oxidative bisulfite pyrosequencing (oxBS-Pyroseq) assay decreased overall or site-specific methylation levels of three of these genes in most cell lines as compared with BS-Pyroseq assay. Similarly, decreased overall or site-specific methylation levels of DAPK, RARβ and RUNX3 genes in laryngeal, gastric and thyroid cancer and their matched normal tissues, respectively, were also found by a comparison between these two techniques, particularly in cancerous tissues. In addition, by using this combined strategy and hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) assay, we demonstrated that TET1 up-regulated DAPK expression through promoter demethylation. Collectively, this strategy is easy to establish and accurately discriminates and quantifies 5mC and 5hmC at CpG sites within selected gene promoters.