Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.