The emergence of massively parallel sequencing (MPS) technologies has revolutionized genetic data generation in many disciplines, and investigations into their use for human identification purposes are well underway. Recent research has demonstrated that these technologies have the potential to recover complete mitochondrial DNA (mtDNA) profiles from even extensively damaged and degraded evidentiary specimens. Yet before MPS can be used for this purpose in regular forensic practice, population reference databases for the entire mitochondrial genome (mtGenome) — developed to the extremely high standards mandated in forensics — must be available to enable the haplotype frequency estimates that are used to assess the strength of mtDNA evidence. To meet this need, we designed a semi-automated, Sanger-based sequencing workflow that consistently generates forensic-quality mtDNA data, and applied the strategy to produce 588 mtGenome haplotypes spanning three U.S. population groups (African American, U.S. Caucasian, and U.S. Hispanic). Data generation metrics demonstrated that large mtDNA fragments can routinely be recovered from very low DNA quantity samples in high-throughput fashion, and indicated the probable success rates of the PCR approach as an enrichment strategy for targeted mtGenome typing of forensic specimens by MPS. Analyses of the data established empirically the increased mtDNA lineage resolution that can be achieved with full mtGenome typing: 90.3% to 98.8% of the haplotypes were unique per population, representing an improvement of 7.7% to 29.2% over mtDNA control region sequencing alone. Maternal biogeographic ancestry proportions inferred from each population sample indicated that the datasets are as representative as the mtDNA control region databases on which haplotype frequency estimates in forensics presently rely. Examination of the data in combination with other recent studies permitted the greatest insight to date into the incidence and distribution of heteroplasmy in the mtDNA coding region, and comparisons of heteroplasmy and substitution patterns provided further support for purifying selection as a mechanism of human mtGenome evolution. Overall, the thoroughly vetted haplotypes can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via MPS) may be evaluated, and will provide a solid foundation for mtGenome haplotype frequency estimates for forensic applications.