Ceratocystis fimbriata is a native, soilborne pathogen in South America that causes a lethal wilt disease on a broad range of economically important plants. Ceratocystis wilt on kiwifruit (Actinidia spp.) was first recognized in 2010 in the state of Rio Grande do Sul, Brazil. The genetic variation among kiwifruit isolates was analized to determine if a single introduced strain of the pathogen was responsible for the epidemic or if there was substantial genetic variation in the population, suggesting that the fungus was soilborne and indigenous to the region. We used 14 microsatellite (simple sequence repeat, SSR) markers to identify 18 genotypes of C. fimbriata among 76 isolates from eight kiwifruit farms. The 18 genotypes clustered into three groups based on UPGMA analysis of the microsatellite alleles. The largest group comprised 60 isolates of 11 closely-related microsatellite genotypes obtained from seven of the eight farms. These genotypes appeared to have originated from a single farm that had supplied cuttings for grafting to the other farms. The population of the pathogen from the farm that supplied the cuttings had the highest level of genotypic diversity and relatively high gene diversity, suggesting that this source population represented an indigenous, soilborne population. Phylogenetic analyses of the DNA sequences of the mating type locus (including portions of MAT1-1-2 and MAT1-2-1) placed the isolates into three groups, corresponding to the three microsatellite groups. Most of the isolates, including all the tested isolates from the farm that supplied the cuttings, had mating type gene sequences that were distinct from other Brazilian populations of C. fimbriata. A second group comprised isolates from one farm that had mating type gene sequences typical of Mata Atlântica (Rio de Janeiro and Sao Paulo) populations of C. fimbriata on Colocasia esculenta and Mangifera indica. Three farms purchased kiwifruit plants or rootstocks from commercial nurseries in Brazil as well as scions from the source farm, and some of the isolates from these farms were genetically similar to Eucalyptus isolates of C. fimbriata from Bahia and Minas Gerais, Brazil. The kiwifruit epidemic in Rio Grande do Sul is the southern-most report of C. fimbriata in Brazil, and the primary pathogen population on kiwifruit appears to be indigenous and originated from a single farm that distributed the pathogen in grafting material. In addition, commercial nursery stock was also implicated as sources of C. fimbriata genotypes. The disease is a major limiting factor for kiwifruit production in southern Brazil, and the results suggest that clean planting stock will be important to successful production.