Pycnidial hyperparasites of powdery mildew fungi, often used in biological control experiments, are currently considered as belonging to a single species, Ampelomyces quisqualis. However, RFLP analysis of the nuclear rDNA ITS region in a worldwide collection of Ampelomyces isolates showed the existence of seven RFLP groups. Cultural characters, e.g. growth rate of isolates, correlated with the exhibited restriction patterns. The results suggested that A. quisqualis should be regarded as a problematic species complex. The geographical distribution of genetically distinct isolates showed that (i) isolates belonging to the same RFLP group were present even in different continents in their fungal hosts, and (ii) isolates representing various RFLP groups were found in the same geographical region, in the same species of the Erysiphaceae and on the same host plants. The detected genetic differences could influence the applicability of various isolates in biological control.