When challenged by difficult biological samples, the forensic analyst is far more likely to obtain useful data by sequencing the human mitochondrial DNA (mtDNA). Nextgeneration sequencing (NGS) technologies are currently being evaluated by the Forensic Science Program at Western Carolina University for their ability to reliably detect lowlevel variants in mixtures of mtDNA. The sequence profiles for twenty individuals were obtained by sequencing amplified DNA derived from the mitochondrial hypervariable (HV) regions using Sanger methods. Two-person mixtures were then constructed by mixing quantified templates, simulating heteroplasmy at discrete sites and in defined ratios. Libraries of unmixed samples, artificial mixtures, and instrument controls were prepared using Illumina^® Nextera^® XT and deep-sequenced on the Illumina^®MiSeq™. Analysis of NGS data using a novel bioinformatics pipeline indicated that minor variants could be detected at the 5, 2, 1, and 0.5% levels of detection. Additional experiments which examined the occurrence of sequence variation in hair tissue demonstrates that a considerable amount of sequence variation can exist between hairs and other tissues derived from a single donor.