Mitochondria within human cells contain vast numbers of mitochondrial DNA (mtDNA), which are small, circular, and double-stranded. The proper functions of mtDNA depend totally on specific proteins that are encoded by the nucleus and then imported into mitochondria. Thus instability of mtDNA can stem from the mtDNA itself, or secondarily from abnormalities in nuclear DNA. In this review, we will first introduce mtDNA, including its characteristics, replication, transcription, translation, and the proteins involved in its metabolism, in particular DNA polymerase gamma (POLG), DNA helicase Twinkle (Twinkle), and mitochondrial transcription factor A (TFAM). Secondly, we will stress the importance of mitochondrial nucleoid structures in the protection and facilitation of mtDNA metabolism, and report on the few known protein components of nucleoid, especially Twinkle, TFAM, and the recently discovered ATAD3. Based on this information, mtDNA instabilities will be categorized in accordance with their molecular etiologies, those that are caused by primary defects of mtDNA, and those by secondary effects from abnormalities in nuclear DNA. The former includes large defects or point mutations of mtDNA. The latter involves the nuclear genes of POLG1, Twinkle, ANT1, TK2, dGK, and TP. With the comprehensive categorization in this review, links are provided between the molecular and clinical aspects of mitochondrial DNA diseases. This report should help medical staff understand the complexity of these diseases and encourage them in further investigations. (