Somatic mitochondrial DNA (mtDNA) mutations have been identified in many human cancers, including leukemia. To identify somatic mutations, it is necessary to have a control tissue from the same individual for comparison. When patients with leukemia achieve remission, the remission peripheral blood may be a suitable and easily accessible control tissue, but this approach has not previously been applied to the study of mtDNA mutations. We have developed and validated a next-generation sequencing approach for the identification of leukemia-associated mtDNA mutations in 26 chronic myeloid leukemia patients at diagnosis using either nonhematopoietic or remission blood samples as the control. The entire mt genome was amplified by long-range PCR and sequenced using Illumina technology. Variant caller software was used to detect mtDNA somatic mutations, and an empirically determined threshold of 2% was applied to minimize false-positive results because of sequencing errors. Mutations were called against both nonhematopoietic and remission controls: the overall concordance between the two approaches was 81% (73/90 mutations). Some discordant results were because of the presence of somatic mutations in remission samples, because of either minimal residual disease or nonleukemic hematopoietic clones. This method could be applied to study somatic mtDNA mutations in leukemia patients who achieve minimal residual disease, and in patients with nonhematopoietic cancers who have a matched uninvolved tissue available.