Salix babylonica L. is a popular ornamental and ecological tree species. In this study, the results of pathogenicity tests showed that the Botryosphaeria dothidea isolates JXL1-3, JXL1-5, JXL1-11, L1-3, L2-2, NFS1, NFS2, NFS5 and WLS1 were pathogens causing branch blight on S. babylonica. Meanwhile, the B. dothidea isolates JXL1-11, L1-3, NFS1 and WLS1 were found to be pathogenic to two other willows, S. chaenomeloides and S. suchowensis. The biological characteristics of B. dothidea isolates JXL1-11, L1-3, NFS1 and WLS1 were investigated under different conditions, including culture media, pH, temperatures, carbon/nitrogen sources and light. It was found that potato dextrose agar (PDA) medium was the most optimal medium for the B. dothidea isolates JXL1-11, L1-3, NFS1 and WLS1 mycelial growth, the suitable pH was 4, and the optimal temperatures were 25°C–30°C. The optimal carbon sources were glucose and sucrose, and the optimal nitrogen sources were ammonium sulfate and peptone. The optimal light condition for inducing sporulation was dark + ultraviolet light. This study provides a fundamental basis for the prevention and control the branch blight on willows and further studies.