Our laboratory has developed a protocol designed to generate whole mitochondrial genome (mt-genome) sequencing data from robust DNA samples, such as those obtained from buccal swabs and blood stains. This approach uses a long PCR amplification technique with two overlapping primer sets, covering the entire human mt-genome. Pooled amplicons are prepared with the Nextera XT™ DNA Sample Preparation Kit and rapidly sequenced on the MiSeq system, generating high-throughput sequencing data that can be quickly analyzed using the on-board software as well as third-party software tools. Data quality is sufficient to easily determine the presence of expected polymorphisms in the samples. In addition, the deep sequencing results on carefully prepared mixtures are able to detect the presence of minor variants at the expected positions, providing a heightened level of detection of minor sequence variants. Results from single source and mixed DNA samples underscore the value and sensitivity of Illumina sequencing data, relative to Sanger sequencing, in providing increased resolution of forensic mtDNA samples.