Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High-resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301–658 bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1 hr.