Management of the patient with oral epithelial dysplasia depends on the ability to predict malignant transformation. Histologic grading of this condition fails in this regard and is also subject to interpathologist and intrapathologist variability. This study uses longitudinal clinical samples to explore the prognostic value of a previously validated panel of methylation biomarkers in a cohort of patients with histologically proven oral dysplasia. Methylation enrichment pyrosequencing assays were used to provide the sensitivity of traditional methylation-specific PCR with the additional specificity advantages of a subsequent confirmatory sequencing reaction. In 57% (8 of 14) patients with a lesion that transformed to oral squamous cell carcinoma, 26% (26 of 100) of longitudinal samples collected over > or =3 years showed p16 methylation. Only 1% (2 of 184) of samples from 8% of patients (2 of 24) not undergoing malignant transformation within 3 years had p16 methylation. Both of these samples with p16 promoter methylation were the most recently collected and the patients remain under continuing clinical review. Promoter methylation of MGMT, CYGB, and CCNA1 did not correlate with malignant progression. We thus conclude that methylation of the p16 gene promoter shows promise as a predictor for malignant transformation (Fisher's exact, P = 0.002) in a subset of patients.