Species in the fungal family Botryosphaeriaceae are latent pathogens on woody trees. These fungi often have a wide host range, which can include native and introduced hosts in an area. Multi-locus DNA sequence identification on a recent collection of Botryosphaeriaceae from Eucalyptus grandis and Syzygium cordatum trees in South Africa revealed cross-infectivity of several species, novel host associations and new country reports. Neofusicoccum eucalyptorum, Neofusicoccum kwambonambiense, Neofusicoccum parvum, Neofusicoccum australe and Lasiodiplodia pseudotheobromae were identified from both tree species, with L. pseudotheobromae and N. eucalyptorum isolated for the first time from S. cordatum, similar to N. kwambonambiense from Eucalyptus. This also represents the first report of L. pseudotheobromae from South Africa. Botryosphaeriaceae species on Eucalyptus species and S. cordatum are fairly well known from South Africa. However, this study revealed new associations, indicating that conclusions should not be generalized and that more intensive sampling from different areas and over time is likely to reveal distinct species and host association patterns. ; The National Research Foundation (NRF), members of the Tree Protection Co-operative Programme (TPCP) and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB), South Africa. Open Access funded by SAAB. ; http://www.elsevier.com/locate/sajb ; hb2016 ; Forestry and Agricultural Biotechnology Institute (FABI) ; Genetics