In forensic casework analysis, mitochondrial deoxyribonucleic acid (DNA) often is used when the evidence material contains scarce amounts of DNA. Here, a mitochondrial DNA typing system for D-loop and coding region analysis based on pyrosequencing is described. Pyrosequencing is a real-time, single-tube sequencing-by-synthesis method, in which a cascade of enzymatic reactions yields detectable light. This pyrosequencing system has a higher resolution than the D-loop analysis performed routinely today as it also covers informative positions in the mitochondrial coding region. The system is composed of 16 polymerase chain reaction (PCR) fragments and 24 pyrosequencing reactions with a turn around time for a 96-well plate of less than 3 h after PCR.