Long-lived rodents have become an attractive model for the studies on aging. To understand evolutionary paths to long life, we prepare chromosome-level genome assemblies of the two longest-lived rodents, Canadian beaver (Castor canadensis) and naked mole rat (NMR, Heterocephalus glaber), which were scaffolded with in vitro proximity ligation and chromosome conformation capture data and complemented with long-read sequencing. Our comparative genomic analyses reveal that amino acid substitutions at “disease-causing” sites are widespread in the rodent genomes and that identical substitutions in long-lived rodents are associated with common adaptive phenotypes, e.g., enhanced resistance to DNA damage and cellular stress. By employing a newly developed substitution model and likelihood ratio test, we find that energy and fatty acid metabolism pathways are enriched for signals of positive selection in both long-lived rodents. Thus, the high-quality genome resource of long-lived rodents can assist in the discovery of genetic factors that control longevity and adaptive evolution. Display omitted •Improved genome assemblies of the naked mole rat and Canadian beaver•Substitutions at disease-causing sites could be adaptive•Unique substitutions in long-lived rodents support stress resistance•Genome resource for studies of long-lived rodents Zhou et al. generate “chromosome-level” genome assemblies for the Canadian beaver and naked mole rat. They characterize genome features and identify common substitutions in long-lived rodents that support enhanced tolerance of cells to DNA damage. The study also provides a valuable genome resource for aging research.