Cellular metabolism is regulated by enzyme complexes within the mitochondrion, the function of which are sensitive to the prevailing temperature. Such thermal sensitivity, coupled with the observation that population frequencies of mitochondrial haplotypes tend to associate with latitude, altitude or climatic regions across species distributions, led to the hypothesis that thermal selection has played a role in shaping standing variation in the mitochondrial DNA (mtDNA) sequence. This hypothesis, however, remains controversial, and requires evidence the distribution of haplotypes observed in nature corresponds with the capacity of these haplotypes to confer differences in thermal tolerance. Specifically, haplotypes predominating in tropical climates are predicted to encode increased tolerance to heat stress, but decreased tolerance to cold stress, than temperate counterparts. We present direct evidence for these predictions, using mtDNA haplotypes sampled from the Australian distribution of Drosophila melanogaster . We show that the ability of flies to tolerate extreme thermal challenges is affected by sequence variation across mtDNA haplotypes, and that the thermal performance associated with each haplotype corresponds with its latitudinal prevalence. The haplotype that predominates at low (subtropical) latitudes confers greater resilience to heat stress, but lower resilience to cold stress, than counterparts predominating at higher (temperate) latitudes. We explore molecular mechanisms that might underlie these responses, presenting evidence that the effects are in part regulated by SNPs that do not change the protein sequence. Our findings indicate that standing genetic variation in the mitochondrial genome can be shaped by thermal selection, and could therefore contribute to evolutionary adaptation under climatic stress.