Genetic variability among sterile cultured single ascospore isolates of Xanthoria parietina, X. calcicola, X. ectaneoides, X. capensis, X. polycarpa and X. resendei was investigated with RAPD-PCR. If available five out of eight ascospores per ascus were analysed. In some samples multispore and mycelial isolates from ascomata were included in the analysis. Ascospore germination rates and phenotypic features such as growth rate, pigmentation and secondary metabolites were uniform in X. parietina sporelings of the same ascus, but varied among the progeny of meiosis in all other species. Phenotypic features correlated with genetic variability. X. parietina revealed polymorphisms among specimens from different worldwide locations. In contrast nine out of ten sets of sibling spores were genetically uniform, with only 2% polymorphism in the remaining set, indicating that X. parietina might be homothallic. X. calcicola, X. ectaneoides, X. capensis, X. polycarpa and X. resendei revealed 9–66% polymorphic loci and therefore are considered heterothallic.