The Botryosphaeriaceae represents an important, cosmopolitan family of latent pathogens infecting woody plants. Recent studies on native trees in southern Africa have revealed an extensive diversity of species of Botryosphaeriaceae, about half of which have not been previously described. This study adds to this growing body of knowledge, by discovering five new species of the Botryosphaeriaceae on Acacia karroo, a commonly occurring native tree in southern Africa. These species were isolated from both healthy and diseased tissues, suggesting they could be latent pathogens. The isolates were characterized based on their morphology and compared to other species for which DNA sequence data are available, in phylogenetic analyses based on the ITS, TEF-1α, β-tubulin and LSU gene regions. The five new species were described as Diplodia allocellula, Dothiorella dulcispinae, Do. brevicollis, Spencermartinsia pretoriensis and Tiarosporella urbis-rosarum. Evidence emerging from this study suggests that many more species of the Botryosphaeriaceae remain to be discovered in the southern Africa. ; Members of the Tree Protection Cooperative Programme (TPCP), the DST/NRF Centre of Excellence in Tree Health Biotechnology (CTHB) and the University of Pretoria, South Africa. ; http://www.cryptogamie.com/pagint_en/recherche/affich_sommaire.php?cnumero=65 ; hb2013