A method for detecting sequence variation of hypervariable segments of the mtDNA control region was developed. The technique uses hybridization of sequence-specific oligonucleotide (SSO) probes to DNA sequences that have been amplified by PCR. The nucleotide sequences of the two hypervariable segments of the mtDNA control region from 52 individuals were determined; these sequences were then used to define nine regions suitable for SSO typing. A total of 23 SSO probes were used to detect sequence variants at these nine regions in 525 individuals from five ethnic groups (African, Asian, Caucasian, Japanese, and Mexican). The SSO typing revealed an enormous amount of variability, with 274 mtDNA types observed among these 525 individuals and with diversity values, for each population, exceeding .95. For each of the nine mtDNA regions significant differences in the frequencies of sequence variants were observed between these five populations. The mtDNA SSO-typing system was successfully applied to a case involving individual identification of skeletal remains; the probability of a random match was approximately 0.7%. The potential useful applications of this mtDNA SSO-typing system thus include the analysis of individual identity as well as population genetic studies.