Sequence variation in hypervariable regions HV1 and HV2 in the control region of mitochondrial DNA (mtDNA) was studied in 100 unrelated Japanese individuals living in Miyazaki Prefecture (in southern Japan) by PCR amplification and direct sequencing. PCR and sequencing primer sets designed for the C-stretch region at around position 16189 in HV1 and position 310 in HV2 were used. Sequence comparison revealed the existence of 90 different haplotypes in 120 variable positions, and of these 82, were unique, 6 were observed twice and 2 were observed three times. Genetic diversity was estimated at 0.999 and the probability of two randomly selected sequences matching (Random Match Probability, RMP) was 1.24%. The C-stretch region in HV1 was observed in 35% of individuals. The average number of nucleotide differences was 9.61 for HV1 and HV2. The majority of sequence variations were substitutions, particularly transitions from thymidine to cytosine (42.6%). Sequence heteroplasmy was not found in this study. Based on the observed polymorphic sites in HV1 and HV2, haplogroups D4 (D4a), M7a, M7b, N9a were the most commonly observed clusters. The resulting data were compared with some existing Japanese mtDNA databases. The present data contribute to expansion of the Japanese mtDNA database, particularly the database for Miyazaki Prefecture. These results show that an mtDNA database is very useful in forensic examination for identification of individuals.