Evolutionary senescence theory predicts that genetically isolated populations historically subjected to low rates of environmentally-imposed mortality will ultimately evolve senescence that is retarded in relation to that of populations historically subjected to higher mortality rates. This prediction was evaluated in the field by comparing three general measures of senescence—age-specific mortality rate acceleration, age-related reproductive output and tail tendon collagen denaturation rate—in two radiocollared Virginia opossum (Didelphis virginiana) populations, one a mainland control and the other an insular population having a four-to five-thousand-year history of reduced exposure to predators. In comparison with those on the mainland, insular female opossums were found to exhibit greater survivorship and reduced litter sizes at all ages, as well as slower acceleration of age-specific mortality. Also, island females in their second reproductive year failed to show the senescent reduction in pouch young growth rate seen in mainland animals. Island opossums also manifested slower ‘ageing’ of tail tendon fibres, a generalized measure of physiological ageing. All these results are consistent with evolutionary senescence theory. Various environmental explanations (parasitism, disease, lessened food availability) for this populational difference are evaluated by physiological measures. No evidence for an environmental explanation is found.