The Radiation Therapy Oncology Group 98-11 clinical trial demonstrated the superiority of standard 5-fluorouracil/mitomycin-C over 5-fluorouracil/cisplatin in combination with radiation in the treatment of anal squamous cell cancer. Tumor size (>5 cm) and lymph node metastases are associated with disease progression. There may be key molecular differences (eg, DNA methylation changes) in tumors at high risk for progression. The objectives of this study were to determine whether there are differences in DNA methylation at individual CpG sites and within genes among locally advanced anal cancers, with large tumor size and/or nodal involvement, compared with those that are less advanced. This was a case-case study among 121 patients defined as high risk (tumor size >5 cm and/or nodal involvement; n = 59) or low risk (≤5 cm, node negative; n = 62) within the mitomycin-C arm of the Radiation Therapy Oncology Group 98-11 trial. DNA methylation was measured using the Illumina HumanMethylation450 Array. The study was conducted in a tertiary care cancer center in collaboration with a national clinical trials cooperative group. The patients consisted of 74 women and 47 men with a median age of 54 years (range, 25-79 years). DNA methylation differences at individual CpG sites and within genes between low- and high-risk patients were compared using the Mann-Whitney test (p < 0.001). A total of 16 CpG loci were differentially methylated (14 increased and 2 decreased) in high- versus low-risk cases. Genes harboring differentially methylated CpG sites included known tumor suppressor genes and novel targets. This study only included patients in the mitomycin-C arm with tumor tissue; however, this sample was representative of the trial. This is the first study to apply genome-wide methylation analysis to anal cancer. Biologically relevant differences in methylated targets were found to discriminate locally advanced from early anal cancer. Epigenetic events likely play a significant role in the progression of anal cancer and may serve as potential biomarkers.