The combination of an enhanced DNA extraction technique, whole genome amplification of the DNA extracts, multiplexed PCR amplification reactions around the mtDNA genome, and direct sequencing of the DNA samples on the Illumina MiSeq instrument resulted in mtDNA sequence information from hair shafts that matched that found in blood and buccal extracts from the same donors. Further developmental research and validation based on this approach and the data obtained should result in a significant improvement over current forensic DNA typing procedures. In this effort, researchers evaluated two newly emerging methods of DNA sequence analysis in order to obtain massively parallel mtDNA sequence information (deep sequencing) from hair, buccal, and blood samples. The expanded information revealed that once this new technology is implemented into casework practice, interpretational change in forensic mtDNA analysis, reflecting the amounts of information that are produced, are necessary. Deep sequencing is a window into a level of variation that is currently under-appreciated in forensic casework. Researchers found that the general level of sequence heteroplasmy present in hair shaft samples, compared to blood and buccal samples, is heightened, but not to a level that would undermine the utility of mtDNA sequencing of hair shaft samples in a forensic context. Using the kit Nextera-XT from Illumina, Inc. enabled researchers to directly process any double-stranded DNA, including amplicons, for deep sequencing in a much simpler and cost-effective manner. 21 figures, 35 tables, and extensive references