For many years it has been assumed that the vast majority of mitochondrial genomes of a single individual are identical, both in the same tissue and within different tissues. Incidences of heteroplasmy (i.e., the occurrence of two or more codominating types of molecules within the mitochondrial DNA population of the same individual) were thought to be extremely rare. This study strongly supports the thesis that heteroplasmy is a principle, rather than an exception, in mitochondrial DNA genetics. During direct sequencing of the first hypervariable segment of the human mitochondrial control region (HV1) in 100 single hair roots obtained from 35 individuals, 24 different heteroplasmic positions were identified. Unusually high levels of heteroplasmy (up to six positions in the HV1 region) were encountered in two individuals. Two individuals related in maternal lineage shared the same heteroplasmic positions. Moreover, highly variable levels of heteroplasmy were observed even among roots from the same individual. The most probable mechanisms involved in generating so many mismatches are mutations occurring presumably in the female germline, followed by differential segregation of mitotypes during the development of individual hairs. Generally, heteroplasmy complicates sequence comparisons in mitochondrial DNA testing performed for forensic purposes, but in some cases it can substantially increase the discriminating power of the analysis.