<jats:title>ABSTRACT</jats:title><jats:p>The variation in the mutational spectrum of the mitochondrial genome (mtDNA) among species is not well understood. Recently, we demonstrated an increase in A>G substitutions on a heavy chain (hereafter A<jats:sub>H</jats:sub>>G<jats:sub>H</jats:sub>) of mtDNA in aged mammals, interpreting it as a hallmark of age-related oxidative damage. In this study, we hypothesized that the occurrence of A<jats:sub>H</jats:sub>>G<jats:sub>H</jats:sub>substitutions may depend on the level of aerobic metabolism, which can be inferred from an organism’s body temperature. To test this hypothesis, we used body temperature in endotherms and environmental temperature in ectotherms as proxies for metabolic rate and reconstructed mtDNA mutational spectra for 1350 vertebrate species. Our results showed that temperature was associated with increased rates of A<jats:sub>H</jats:sub>>G<jats:sub>H</jats:sub>and asymmetry of A<jats:sub>H</jats:sub>>G<jats:sub>H</jats:sub>in different species of ray-finned fishes and within geographically distinct clades of European anchovy. Analysis of nucleotide composition in the most neutral synonymous sites of fishes revealed that warm-water species were expectedly more A-poor and G-rich compared to cold-water species. Finally, we extended our analyses to all vertebrates and observed higher A<jats:sub>H</jats:sub>>G<jats:sub>H</jats:sub>and increased asymmetry of A<jats:sub>H</jats:sub>>G<jats:sub>H</jats:sub>in warm-blooded (mammals and birds) compared to cold-blooded (Actinopterygii, amphibia, reptilia) vertebrate classes. Overall, our findings suggest that temperature, through its influence on metabolism and oxidative damage, shapes the mutational properties and nucleotide content of the mtDNA in all vertebrates.</jats:p>