AbstractWhen applying genome‐wide sequencing technologies to disease investigation, it is increasingly important to resolve sequence variation in regions of the genome that may have homologous sequences. The human mitochondrial genome challenges interpretation given the potential for heteroplasmy, somatic variation, and homologous nuclear mitochondrial sequences (numts). Identical twins share the same mitochondrial DNA (mtDNA) from early life, but whether the mitochondrial sequence remains similar is unclear. We compared an adult monozygotic twin pair using high‐throughput sequencing and evaluated variants with primer extension and mitochondrial preenrichment. Thirty‐seven variants were shared between the twin individuals, and the variants were verified on the original genomic DNA. These studies support highly identical genetic sequence in this case. Certain low‐level variant calls were of high quality and homology to the mtDNA, and they were further evaluated. When we assessed calls in preenriched mtDNA templates, we found that these may represent numts, which can be differentiated from mtDNA variation. We conclude that twin identity extends to mtDNA, and it is critical to differentiate between numts and mtDNA in genome sequencing, particularly as significant heteroplasmy could influence genome interpretation. Further studies on mtDNA and numts will aid in understanding how variation occurs and persists.