Mitochondrial DNA (mtDNA) analysis is a useful tool to analyze forensic samples. MtDNA exists as numerous molecules in a single cell in contrast to nuclear DNA, therefore mtDNA analysis can provide successful results in analyzing minute DNA samples. In our current PCR protocol of mtDNA analysis, hypervariable region 1 (HV1) and hypervariable region 2 (HV2) are divided into 5 regions (region A to E), and each region is amplified separately. It requires labor and long PCR time. In this paper, we study multiplex PCR of mtDNA and high-speed PCR using high-speed DNA polymerase to reduce labor and time of mtDNA analysis.