Ceratocystis pirilliformis was first isolated from wounds on Eucalyptus nitens in Australia and subsequently found in a similar niche on E. grandis in South Africa. Artificial inoculation studies under field conditions in South Africa resulted in bark lesions and sap-stain, suggesting that the fungus is a pathogen of potential importance to forestry in South Africa. Because Eucalyptus spp. are native to Australia and C. piriliformis was first found there in the absence of disease it has been assumed that the fungus is native to Australia. The aim of this study was to expand the base of knowledge regarding the distribution and population diversity of C. pirilliformis in South Africa. Wounds were examined on Eucalyptus spp. growing in seven of the most important forestry areas in South Africa. PCR-based microsatellite markers, developed for the closely related tree pathogen C. fimbriata sensu lato were used to assess the diversity of the isolates collected. Ceratocystis pirilliformis was found in four out of seven areas surveyed, substantially expanding its known distribution in South Africa. Of the 27 available microsatellite markers, 18 amplified the desired loci of C. pirilliformis and of these, seven were polymorphic. Measures of genetic diversity based on gene diversity, genotypic diversity as well as allelic richness indicated that the isolates from South Africa has a low level of genetic diversity and that the fungus is most likely not native to the country. Inclusion of available Australian isolates showed much higher diversity for C. pirilliformis in that country.