Cytospora spp. (anamorphs of Valsa spp.) are common inhabitants of woody plants and they include important stem and branch canker pathogens. Isolates of these fungi were collected from diseased and healthy trees in South Africa. They were identified based on morphology and DNA sequence homology of the intertransgenic spacer ribosomal DNA. South African isolates were compared with isolates collected in other parts of the world, and they represented 25 genetically distinct sequences residing within the populations of 13–14 known species and three unique lineages. Several species are new records for South Africa, doubling previous reports of these fungi from the country. Similarities between South African isolates of Cytospora from non-native Eucalyptus, Malus, Pinus, Populus, Prunus and Salix species and isolates from Australia, Europe or America suggest that the fungal pathogens were imported with their hosts as endophytes. Isolates from indigenous Olea and Acacia appear to represent native populations. Host shifts were evident, including populations on Eucalyptus that also occurred on Mangifera, Populus, Sequoia, Tibouchina and Vitex. Isolates related to Valsa kunzei represent the first report of a Cytospora species on the widely cultivated timber tree, Pinus radiata. An identification key to Cytospora species in South Africa is included.