As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.