<p>Aging is a critical risk factor for cancer development with genetic instability presenting as a common hallmark. Alternative DNA-forming sequences are endogenous sources of genetic instability that are enriched at mutation hotspots in cancer genomes. Using a transgenic mutation-reporter mouse model containing an H-DNA-forming sequence from a mutation hotspot in Burkitt lymphoma, we demonstrate tissue-specific effects of aging on DNA structure-induced mutagenesis, with H-DNA mutation frequencies increasing in spleen and liver and decreasing in brain tissues. DNA sequencing revealed a correlation of increasing large deletions with increasing mutation frequencies. Further, we observed tissue-specific modulation of mechanisms of H-DNA processing, including decreased nucleotide excision repair activity in aged brain tissues and increased cleavage activity on H-DNA structures in aging spleen tissues. Together, these findings provide significant insights into the relationship between aging and cancer-associated genetic instability, aiding in the delineation of the underlying mechanisms of age-associated cancer development.</p>