We performed a global methylation profiling assay on 1505 CpG sites across 807 genes to characterize DNA methylation patterns in pancreatic cancer genome. We found 289 CpG sites that were differentially methylated in normal pancreas, pancreatic tumors and cancer cell lines. We identified 23 and 35 candidate genes that are regulated by hypermethylation and hypomethylation in pancreatic cancer, respectively. We also identified candidate methylation markers that alter the expression of genes critical to gemcitabine susceptibility in pancreatic cancer. These results indicate that aberrant DNA methylation is a frequent epigenetic event in pancreatic cancer; and by using global methylation profiling assay, it is possible to identify these markers for diagnostic and therapeutic purposes in this disease.