Chile is one of the major exporters of fresh apples worldwide with a production area of 37,000 ha. The fungal diseases are one of the major problems for fresh apple production, and in recent years, symptoms of Botryosphaeria canker and dieback of arms and twigs have become an important threat for apple growers in Chile. However, a systematic study of the importance of Botryosphaeria canker and dieback in the field has not yet been performed. Therefore, field surveys were conducted throughout the main apple-growing regions to determine the prevalence and to identify the Botryosphaeriaceae species associated with cankers and dieback in apple trees in Chile. A total of 115 samples of symptomatic branches or twigs were plated on PDA plus antibiotics and Igepal. Cultures were identified morphologically and molecularly using the ITS region of rDNA, a portion of the ?­tubulin and a part of EF1­? genes. The results allowed to determine a prevalence between 8 and 40% of canker and dieback in apple orchards. Based on morphologic and phylogenetic analyses, we identified three species belonging to Botryosphaeriaceae family: Diplodia seriata, Neofusicoccum arbuti and Lasiodiplodia theobromae. All the isolates were pathogenic on 1-years old dormant cuttings and on twigs in the orchard. Additionally, these isolates caused rot lesions on inoculated apple fruits and were sensitive in vitro to several fungicides. The fungus D. seriata was the most frequent Botryosphaeriaceae specie obtained from apple trees with canker and dieback in Chile.