We sequenced the complete 16.5-kb mitochondrial genome (mtDNA) in 15 pancreatic cancer cell lines and xenografts. Homoplasmic mtDNA somatic mutations and novel variants were identified in nearly all samples. Southern blot analysis and direct sequencing of mutation sites showed that the intracellular mass of mtDNA was greatly (6–8-fold) increased in pancreatic cancer cells in relation to corresponding normal cells;this property accounted for and greatly facilitated the identification of these mutations among the dense desmoplastic host reaction characteristic of primary pancreatic cancers. Structural characteristics and mathematical modeling of the evolution of mtDNA mutations suggested that many of the mutations identified might represent a random evolution of homoplasmic variants, rather than necessarily being a product of selective pressures. Complete sequencing of the nuclear MnSOD gene, which protects cells from the mitogenic and toxic effects of oxygen radicals, did not reveal any mutations. Nevertheless, the nearly ubiquitous prevalence and high copy number of mtDNA mutations suggest that they be considered of promising clinical utility in diagnostic applications.