Epigenetic changes have been implicated in the pathogenesis of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Prior efforts have primarily examined regional promoter hypermethylation as a silencer of tumor suppressor gene expression. To analyze the global state of methylation in the HNSCC genome, we utilize pyrosequencing of repetitive elements (LINEs) to compare the state of global methylation in HNSCC to normal aerodigestive mucosa. 137 samples (119 HNSCC tumors and 18 normal mucosal tissues) were digested to extract DNA and subjected to bisulfite treatment. Treated DNA was amplified using PCR primers for the repetitive LINEs sequence and produced a heterogeneous sample of products, from many genomic loci. These products were pyrosequenced to quantitatively evaluate their global genomic methylation status. HNSCC specimens showed global hypomethylation, with a mean level of genomic methylation of 46.8% methylated with a standard deviation of 9.0. Conversely, the normal upper airway mucosa had a global methylation level of 54.0 and a standard deviation of 4.6 (Mann–Whitney p value < 0.001). The tumor specimens also showed an increasing degree of hypomethylation associated with advanced tumor stage (ANOVA p-value of 0.003). About 67% of HNSCC's are globally hypomethylated when evaluated against the minimum level of methylation in the normal mucosal specimens. Degree of global hypomethylation was associated with smoking history, alcohol use and stage in univariate analysis (p-value 0.02), however, only HNSCC diagnosis remained significant on multivariate analysis. Despite the presence of regional promoter hypermethylation, HNSCC demonstrates global genomic hypomethylation. The effects of stage, alcohol use and smoking on global hypomethylation were not independently significant. © 2007 Wiley-Liss, Inc.