Cancer arises due to the accumulation of DNA modifications that give cells a selective growth advantage. One common DNA modification is promoter hypermethylation associated with loss of expression of a tumor suppressor gene. The methylation status of a specific sequence or the pattern of methylation across the genome can be readily measured, and these sequences and analytical methods are being rapidly developed for molecular diagnostic applications. Detection of certain methylation events can be used for early detection of tumors, and analysis of patterns of methylation across the genome might provide information on disease subtype, aggressiveness, and treatment response. DNA methylation-based molecular diagnostic assays are particularly attractive because of the stability of the target analyte (DNA) and the potential sensitivity of the assays. As the field matures, methylation-based assays will make a major contribution to the field of molecular diagnostics, providing tools to fill unmet needs in current diagnostic and treatment plans for many types of cancer.