Mitochondrial DNA (mtDNA) is a powerful tool for forensic casework when the resource is highly putrefied or very limited. It is important that mtDNA sequence databases continue to be generated and published, to extend mtDNA typing capability to additional populations and to increase the size of existing databases. The aim of this study was to analyze hypervariable region 1 (HV1) of mtDNA and establish a new database of mtDNA in Japanese population. Three hundred and ninety-four unrelated Japanese individuals were investigated. They were classified into 193 haplotypes based on HV1 sequence. Nucleotide substitutions were clustered in several positions and were 79% at 16,223, 50% at 16,362, and 29% at 16,189. Transition accounts for 81% of all the mutations and especially transition from cytosine to thymine (33%) was the most frequent substitution. 16,223–16,362 was observed in 34 individuals, following 16,129–16,223–16,362 in 29 individuals, and haplotype 16,223–16,278–16,362 in 14 individuals. Genetic diversity and random match probability in this population were estimated to 0.987% and 1.95%, respectively. In conclusion, HV1 of mitochondria sequence has a high discrimination power, but in addition to HV1, analysis of other region such as HV2 and HV3 are required in certain cases.