Unusual and extensive dieback of European hop hornbeam (Ostrya carpinifolia) has been observed in western Slovenia and northern Italy in recent years, when extreme drought and higher temperatures were recorded. A preliminary study identified Botryosphaeria dothidea as a potential causative agent of the dieback. Further characterization of intra- and interspecies diversity of Botryosphaeriaceae collected from O. carpinifolia and other tree species in the affected area was achieved based on anamorph morphology, sequence data from the ITS rDNA and EF1-α, PCR–RFLP analysis and AFLP profiles. The majority of the isolates analyzed were identified as B. dothidea, and in vitro pathogenicity tests re-confirmed the fungus to be an important cause of the disease. Insight into the B. dothidea population, diversity based on AFLP markers indicates that the dieback observed in O. carpinifolia is probably associated with a heterogenous population of B. dothidea, which emerged from a latent state in response to changed climate conditions. Isolates with Dothiorella-like conidia were also identified during the survey, but these were collected more rarely and appear to represent undescribed species. Isolates from Dothiorella genus expressed low pathogenicity in pathogenicity tests and proved no impact on the pathogenic ability of B. dothidea.