To elucidate the primary factors shaping mitochondrial DNA (mtDNA) mutagenesis, we reconstructed a deep 192-component mtDNA mutational spectrum using 129,100 polymorphic synonymous mutations from the Cytb gene of 1040 chordate species. We then deconvoluted this spectrum into three key signatures: (i) symmetrical mutations, predominantly C>T and rare transversions, linked to pol-γ’s replication errors; (ii) asymmetrical C>T mutations, indicative of single-stranded DNA damage; and (iii) asymmetrical A>G mutations, also resulting from single-stranded DNA damage but particularly influenced by metabolic and age-specific mitochondrial environment. Interestingly, the two asymmetrical signatures collectively surpass pol-γ mutations, indicating that damage is the primary factor in mtDNA mutagenesis over replication. The diverse contribution of all three signatures across different species and human tissues provides insights into a range of physiological and metabolic traits. This mtDNA spectrum-aware methodology enhances phylogenetic inferences, deepens our understanding of species-specific mutational and selection processes, and offers insights into the dynamics of mutations in human mtDNA.