AbstractHead and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with more than half a million people being diagnosed with the disease annually. Within the last 2 decades, the human papillomavirus (HPV) has been found to be associated with this malignancy. More recently, HPV‐infected HNSCC has been found to exhibit higher levels of global DNA methylation. In a recent study, we identified five tumor suppressive genes (IRX1, EBF3, SLC5A8, SEPT9, and FUSSEL18) as frequently methylated in HNSCC biopsies using a global methylation analysis via restriction landmark genomic scanning. In this study, we verify these genes as valid methylation markers in two separate sets of HNSCC specimens. By using the available clinical information linked to the patient specimens, we found a strong association between promoter methylation of FUSSEL18, IRX1, and EBF3 and prior radiation therapy (P < 0.0001) irrespective of HPV status. Also, promoter methylation of FUSSEL18 and SEPTIN9 was found to correlate significantly with exposure to alcohol and tobacco (P = 0.021). Importantly, in this study, we preliminarily show a trend between HPV16 positivity and specific target gene hypermethylation of IRX1, EBF3, SLC5A8, and SEPT9. If replicated in a larger study, the HPV status may be a patient selection biomarker when determining the most efficacious treatment modality for these different subsets of patients (e.g., inclusion or exclusion of epigenetic therapies). Equally notable and independent of HPV status, hypermethylation of the promoters of a subset of these genes in recurrences especially in the setting of prior radiation or in the setting of alcohol and tobacco use might help guide adjunctive inclusion or exclusion or epigenetic therapy. © 2009 Wiley‐Liss, Inc.