Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus from Africa showed that some isolates collected from hardwoods were genetically distinct from O. quercus. In the present study we compared these African isolates with authentic O. quercus isolates, including the neo-type of the species from oak in France, by evaluating morphological characters, growth in culture, mating compatibility, and DNA sequence data. The isolates from Africa exhibited substantial variability in culture and were morphologically similar to O. quercus. Phylogenetic analyses of the ITS, β-tubulin and Translation Elongation Factor 1-α gene regions confirmed that the African group represents a distinct species in the hardwood lineage of the O. piceae-complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates predominantly mated with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage is described here as a new species, O. tsotsi sp. nov., and is characterized based on morphological characteristics, growth in culture and sequence comparisons.