Members of Botryosphaeriales are ecologically diverse, but most commonly associated with leaf spots, fruit and root rots, die-back or cankers of diverse woody hosts. Based on morphology and DNA sequence data, the Botryosphaeriales have to date been shown to contain eight families, with an additional two, Endomelanconiopsisaceae (Endomelanconiopsis) and Pseudofusicoccumaceae (Pseudofusicoccum) being newly described in this study. Furthermore, Oblongocollomyces is introduced as new genus, while Spencermartinsia is reduced to synonymy under Dothiorella. Novel species include Diplodia pyri (Pyrus sp., the Netherlands), Diplodia citricarpa (Citrus sp., Iran), Lasiodiplodia vitis (Vitis vinifera, Italy), L. sterculiae (Sterculia oblonga, Germany), Neofusicoccum pistaciarum (Pistacia vera, USA), N. buxi (Buxus sempervirens, France), N. stellenboschiana (Vitis vinifera, South Africa), and Saccharata hawaiiensis (Protea laurifolia, Hawaii). New combinations are also proposed for Camarosporium pistaciae (associated with fruit rot of Pistacia vera) in Neofusicoccum, and Sphaeria gallae (associated with galls of Quercus) in Diplodia. The combination of large subunit of the nuclear ribosomal RNA gene (LSU)-rpb2 proved effective at delineating taxa at family and generic level. Furthermore, rpb2 also added additional resolution for species delimitation, in combination with ITS, tef1 and tub2. In this study we analysed 499 isolates, and produce an expanded phylogenetic backbone for Botryosphaeriales, which will help to delimit novelties at species, genus and family level in future.