Botryosphaeria dieback (BD) occurs in young and old plants. In the field, the prevalence and severity of the disease increase proportionally with the age of vineyards. Among the pathogens that cause BD, Diplodia seriata is the most prevalent species in Chile and other countries with a Mediterranean climate. To date, no information is available on the susceptibility of adult wood to infection by this pathogen since most of the pathogenicity tests have been carried out on 1- or 2-year-old shoots or detached canes. Therefore, a pathogenicity test was carried out on plants under field conditions, with inoculations in 1-year-old shoots and 2- and 10-year-old wood in grapevine cv. Cabernet Sauvignon. A pathogenicity test was carried out with two isolates of D. seriata. The results for the plants show that D. seriata was significantly more aggressive on the 10-year-old than on the one- or two-year-old tissue, where the lesions were 4.3 and 2.3 cm on average, respectively. These results were compared with the lesions obtained from two-year-old canes after the isolates were activated in grape berries. Also, the Chilean isolates of D. seriata were compared phylogenetically with those from other countries, and no major differences were found between them. Our results are consistent with the damage observed in the field, contributing to the knowledge of the epidemiology of this disease in Mediterranean climates. In the future, the effect observed in cv. Cabernet Sauvignon with D. seriata on virulence at different tissue ages should be tested for other BD-causing agents and wine varieties.