Mitochondrial DNA (mtDNA) mutagenesis remains poorly understood despite its crucial role in disease, aging, and evolutionary tracing. In this study, we reconstructed a comprehensive 192-component mtDNA mutational spectrum for chordates by analyzing 118,397 synonymous mutations in the CytB gene across 1,697 species and five classes. This analysis revealed three primary forces shaping mtDNA mutagenesis: (i) symmetrical, replication-driven errors by mitochondrial polymerase (POLG), resulting in C >
T and A >
G mutations that are highly conserved across classes
(ii) asymmetrical, damage-driven C >
T mutations on the single-stranded heavy strand with clock-like dynamics
and (iii) asymmetrical A >
G mutations on the heavy strand, with dynamics suggesting sensitivity to oxidative damage. The third component, sensitive to oxidative damage, positions mtDNA mutagenesis as a promising marker for metabolic and physiological processes across various classes, species, organisms, tissues, and cells. The deconvolution of the mutational spectra into mutational signatures uncovered deficiencies in both base excision repair (BER) and mismatch repair (MMR) pathways. Further analysis of mutation hotspots, abasic sites, and mutational asymmetries underscores the critical role of single-stranded DNA damage (components ii and iii), which, uncorrected due to BER and MMR deficiencies, contributes roughly as many mutations as POLG-induced errors (component i).