There is strong evidence that mitochondria once existed as free-living bacteria, which were taken up by primitive ancestors of eukaryotic cells in an arrangement termed endosymbiosis. The primitive host cell, which had an organized nucleus, provided a ready source of energy-rich nutrients to the mitochondrion, and the mitochondrion provided the cell with a means to extract energy using oxygen. This attribute became key to survival, as the primitive atmosphere shifted from reducing to oxidizing. Although the earliest atmosphere was composed primarily of hydrogen, oxygen began to accumulate with the advent of photosynthesis. Like mitochondria, chloroplasts have their own genome and were once free-living bacteria. Thus, early plant cells evolved by serial endosymbiosis: successively engulfing two sorts of bacteria to obtain mitochondria and chloroplasts.

The human mt genome, like those of other eukaryotes, has been vastly reduced through evolutionary time. The free-living ancestor of mitochondria, perhaps similar to a Rickettsia, must have had a complement of at least 850 genes. Over time, genes for functions that could be provided by the host were lost. Also, some genes needed for respiration were transferred to the nucleus. Over millions of years of evolutionary time, this reduction resulted in the small mt chromosome found in eukaryotes.

Besides having similar dimensions, mitochondria and bacteria share several genomic features that demonstrate their common ancestry. Like bacterial chromosomes and plasmids, the mt genome is a circular molecule. A circular configuration was the first mechanism to provide protection from exonucleases, which digest free ends of linear DNA molecules. By contrast, "end caps" of repetitive DNA sequences, the telomeres, protect the linear chromosomes of eukaryotic organisms. Mt and bacterial genomes, also, generally have little noncoding DNA. Genes are usually tightly packed together on the chromosome, with few intergenic regions between genes and few introns within genes. These features are contrary to eukaryotic genes, which are widely dispersed on chromosomes and have numerous introns.