Background: We explored the utility of multiplexed temperature gradient capillary electrophoresis (TGCE) as a screening tool for identifying genetic changes in the human mitochondrial genome. We examined changes in mitochondrial DNA (mtDNA) in nonmelanoma skin cancers (NMSCs), using TGCE to resolve genetic differences contained within the tumors compared with the control DNA. Methods: The entire mtDNA from NMSC tissue samples was amplified in 17 overlapping amplicons averaging 1.1 kb in size. Fourteen of these amplicons were digested with restriction endonucleases into as many as five smaller analyzable fragments. Digested tumor mtDNA amplicons were annealed with digested amplicons from the control DNA to form heteroduplexes in regions of DNA mismatch. TGCE was performed in a 96-well parallel format to detect mtDNA changes in a high-throughput fashion. Results: TGCE resolved heteroduplexes from homoduplexes in singlet reactions and in multiplexed assays. Using a single programmed temperature gradient, we detected 18 of 20 mtDNA changes contained within the specimens. This system was also able to detect a single nucleotide change in a fragment as large as 2 kb. Conclusion: Multiplexed TGCE is a sensitive and high-throughput screening tool for identifying mtDNA variations.