Species of the family Botryosphaeriaceae (Botryosphaeriales) have a worldwide distribution and some species of this family have been recognized as important pathogens on various trees worldwide. In order to identify the Botryosphaeriaceae spp. associated with olive trunk diseases, a survey was conducted on trees in different olive-growing regions in Kerman and Mazandaran provinces. In 2016, wood samples were collected from trunk and branches of trees showing dieback and canker as well as internal wood necrosis in cross sections. Fungal isolations were performed from discolored wood tissues. Small wood segments (5×5 mm) were cut from affected tissues and surface-sterilized in 0.5 % sodium hypochlorite for 2 min followed by two rinses in sterile distilled water and then placed on potato dextrose agar (PDA) amended with streptomycin sulphate. In this study 22 fungal isolates resembling Botryosphaeriaceae species in colony and culture (fast growing, greyish to dark cultures) were obtained from affected trees. To induce sporulation, fungal isolates were also sub-cultured on water-agar with autoclaved pine needles and stored for 2-5 weeks at room temperature under fluorescent white light. All isolates were identified tentatively by means of morphological features of conidia (size, colour and septation) and confirmed by partial sequences of the translation elongation factor 1-α gene (EF-1α) with the primer pairs EF1-728F and EF1-986R. Based on morphological and molecular analysis four species of Botryosphaeriaceae namely, Botryosphaeria dothidea (five isolates), Diplodia seriata (five isolates), Dothirella sarmentorum (eight isolates) and Lasiodiplodia theobromae (four isolates) were isolated and identified from olive trees. This study is the first report of B. dothidea, D. seriata, and L. theobromae from olive trees in Iran. In addition, this study is the first report of Do. sarmentorum associated with yellowing, canker and dieback symptoms of olive trees.