The well-established correlation between global DNA hypomethylation and genomic instability offers one of the mechanistic explanations on how hypomethylation may contribute to carcinogenesis. In our study, levels of 5-methyl-2’-deoxycytidine in oral cancer tissues and microdissected DNA analysed by reverse phase – high performance liquid chromatography (RP-HPLC) revealed a generalized decrease in all oral cancer samples (1.63 ± 0.16) compared to its matched normal counterpart (3.67 ± 0.17; P < 0.001). Validation of global DNA methylation as analysed by immunohistochemistry (IHC) in successive stages of oral cancer progression in tissue sections using antibody to 5-methyl cytosine revealed reduced immunostaining in dysplasia and oral squamous cell carcinomas (OSCC) compared to normal oral mucosal epithelium. Methylation sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) identified several non-canonical CpG rich fragments including promoter region of DOC2B (Double C2 like Domain B) gene spanning -376bp to + 36bp. This was found to be methylated in oral cancer tissues, confirmed by methylation sensitive dimethyl sulfoxide polymerase chain reaction (MS-DMSO-PCR) and validated by bisulfite genome sequencing (BGS). DOC2B promoter and other differentially methylated sequences identified in our study may serve as potential diagnostic markers to delineate human oral cancer and suggests oral cancer pathogenesis to altered epigenetic mechanisms.