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The aetiology of OSCC remains unclear, however, aberrant methylation of CpG island promoters of tumor suppressor genes have been identified as contributory developmental pathways in several cancers. The aim of this study was to determine the presence of RUNX3 gene methylation and how its association with patients' demographic variables such as gender, age, histologic class and tumor location could be of diagnostic value for OSCC. Sixty-seven formalin-fixed paraffin-embedded (FFPE) solid tissue blocks of OSCC, and nine blocks of benign oral lesions of epithelial origin retrieved from the archives of the Department of Oral Pathology, University College Hospital, Ibadan, South-West Nigeria were used for the analyses. Frequency of CpG island methylation in the promoter region of RUNX3 was determined by methylation-specific polymerase chain reaction (MSP). Association between gender, age, tumor location, histologic class and promoter methylation in RUNX3 was assessed with Pearson's X2 test. Overall, 45% (30/67) of OSCC demonstrated methylation in the RUNX3 promoter indicating a high frequency of methylation of the CpG island promoter region of RUNX3. There was no association between gender, age, histologic class and promoter methylation in RUNX3 (P > 0.05), however a significant association was observed between tumor location and promoter methylation of RUNX3 (P < 0.05). Aberrant methylation of the CpG island promoter region of RUNX3 together with tumor location could therefore be critical in the development and diagnosis of OSCC. [ABSTRACT FROM AUTHOR]