Mitochondria are thought to play a central role in aging. In humans, specific naturally occurring mitochondrial genetic variants are overrepresented among centenarians, but only in certain populations; therefore, we cannot tell whether this effect is due solely to mitochondrial genetics or to nuclear-mitochondrial gene complexes, nor do we know the magnitude of the effect in terms we can relate to, such as mean lifespan differences. To examine the effects of natural mitochondrial DNA (mtDNA) variation on lifespan, we need to vary the mitochondrial genotype while controlling the nuclear genotype. Here, nuclear genome replacement is achieved using strains of Drosophila melanogaster bearing multiply inverted 'balancer' chromosomes that suppress recombination, and an isogenic donor strain, thus forcing replacement of entire chromosomes in a single cross while suppressing recombination. Lifespans of wild-type mtDNA variants on the chromosome replacement background vary substantially, and sequencing of the entire protein coding mitochondrial genomes indicates that these lifespan differences are sometimes associated with single amino acid differences. On other nuclear genetic backgrounds, the magnitude and direction of these lifespan effects can change dramatically, and this can be due to changes in baseline mortality risk, rate of aging and/or time of onset of aging. The limited mtDNA variation in D. melanogaster makes it an ideal organism for biochemical studies to link genotype and aging phenotype.