Mitochondrial DNA (mtDNA or mDNA) is the DNA located in organelles called mitochondria, structures within eukaryotic cells that convert the chemical energy from food into a form that cells can use, adenosine triphosphate (ATP). Most other DNA present in eukaryotic organisms is found in the cell nucleus.

Mitochondrial DNA can be regarded as the smallest chromosome, and was the first significant part of the human genome to be sequenced. In most species, including humans, mtDNA is inherited solely from the mother.

The DNA sequence of mtDNA has been determined from a large number of organisms and individuals (including some organisms that are extinct), and the comparison of those DNA sequences represents a mainstay of phylogenetics, in that it allows biologists to elucidate the evolutionary relationships among species. It also permits an examination of the relatedness of populations, and so has become important in anthropology and field biology.

Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today's eukaryotic cells. This theory is called the endosymbiotic theory. Each mitochondrion is estimated to contain 2-10 mtDNA copies. In the cells of extant organisms, the vast majority of the proteins present in the mitochondria (numbering approximately 1500 different types in mammals) are coded for by nuclear DNA, but the genes for some of them, if not most, are thought to have originally been of bacterial origin, having since been transferred to the eukaryotic nucleus during evolution.