Mitochondrial DNA (mtDNA) defects cause debilitating metabolic disorders for which there is no effective treatment. Patients suffering from these diseases often harbour both a wild‐type and a mutated subpopulation of mtDNA, a situation termed heteroplasmy. Understanding mtDNA repair mechanisms could facilitate the development of novel therapies to combat these diseases. In particular, mismatch repair activity could potentially be used to repair pathogenic mtDNA mutations existing in the heteroplasmic state if heteroduplexes could be generated. To date, however, there has been no compelling evidence for such a repair activity in mammalian mitochondria. We now report evidence consistent with a mismatch repair capability in mammalian mitochondria that exhibits some characteristics of the nuclear pathway. A repair assay utilising a nicked heteroduplex substrate with a GT or a GG mismatch in the β‐galactosidase reporter gene was used to test the repair potential of different lysates. A low level repair activity was identified in rat liver mitochondrial lysate that showed no strand bias. The activity was mismatch‐selective, bi‐directional, ATP‐dependent and EDTA‐sensitive. Western analysis using antibody to MSH2, a key nuclear mismatch repair system (MMR) protein, showed no cross‐reacting species in mitochondrial lysate. A hypothesis to explain the molecular mechanism of mitochondrial MMR in the light of these observations is discussed.