A new original method of quantitative estimation of heteroplasmy levels in mitochondrial genome is necessary for determination cut-of values mutant allele in tissues, associated with the emergence and development of pathologies in human organism. This method is based on pyrosequencing of short DNA fragments. It is characterized by high accuracy and good reproducibility and helps to find out crucial differences in the level of heteroplasmy of mitochondrial genome in various organs and tissue fragments from the same individual.