The genus Botryosphaeria includes more than 200 epithets, but only the type species, Botryosphaeria dothidea and a dozen or more other species have been identified based on DNA sequence data. The taxonomic status of the other species remains unconfirmed because they lack either morphological information or DNA sequence data. In this study, types or authentic specimens of 16 “Botryosphaeria” species are reassessed to clarify their identity and phylogenetic position. nuDNA sequences of four regions, ITS, LSU, tef1-α and tub2, are analyzed and considered in combination with morphological characteristics. Based on the multigene phylogeny and morphological characters, Botryosphaeria cruenta and Botryosphaeria hamamelidis are transferred to Neofusicoccum. The generic status of Botryosphaeria aterrima and Botryosphaeria mirabile is confirmed in Botryosphaeria. Botryosphaeria berengeriana var. weigeliae and B. berengeriana var. acerina are treated synonyms of B. dothidea. Botryosphaeria mucosa is transferred to Neodeightonia as Neodeightonia mucosa, and Botryosphaeria ferruginea to Nothophoma as Nothophoma ferruginea. Botryosphaeria foliicola is reduced to synonymy with Phyllachorella micheliae. Botryosphaeria abuensis, Botryosphaeria aesculi, Botryosphaeria dasylirii, and Botryosphaeria wisteriae are tentatively kept in Botryosphaeria sensu stricto until further phylogenetic analysis is carried out on verified specimens. The ordinal status of Botryosphaeria apocyni, Botryosphaeria gaubae, and Botryosphaeria smilacinina cannot be determined, and tentatively accommodate these species in Dothideomycetes incertae sedis. The study demonstrates the significance of a polyphasic approach in characterizing type specimens, including the importance of using of DNA sequence data.