The ability to analyze whole mt genome sequences in degraded forensic samples, as well as mixture samples, will have a major impact on the criminal justice system. The primary goal of this research project was to validate a previously developed and optimized probe capture next-generation sequencing (NGS) assay for sequencing of the entire mitochondrial (mt) genome for forensically challenging samples. The mt genome probe capture NGS assay developed can be used on reference and compromised samples, including highly degraded, mixed, and limited DNA samples often encountered in forensic cases or mass disasters. The specific aims of this project were to: 1) complete the developmental validation of the mt genome probe capture NGS assay; 2) test the performance on highly degraded, limited, and mixed forensic type samples; 3) complete internal validation studies (external collaborators); 4) conduct comparative NGS technology studies; and 5) customize and validate mtDNA analysis tools. The successful validation studies for the mt genome probe capture NGS system developed during the research will provide an alternative method for analysis of mtDNA for application with highly degraded, limited, and mixed DNA samples often encountered in missing person identification, mass disaster cases, and forensic casework.