Isolates of the eyespot pathogen of cereals may be differentiated into two main groups on the basis of a series of cultural and molecular markers. A heterothallic mating system with mating types MAT1-1 and MAT1-2 has already been demonstrated in one group, Tapesia yallundae. A comparable mating system is now described in the other group based on results from in vitro crosses in which the sexual cycle was induced on straw segments incubated at 7 °C or less under nuv or white light for a minimum of 8 months. Recombination between two parental strains was detected using randomly amplified polymorphic DNA (RAPD) molecular markers, and this led to the appearance of novel phenotypes amongst the ascospore progeny including the generation of a new RAPD band not present in either parent. In vitro crossing tests also revealed that the two main groups were sexually incompatible and thus represent genetically isolated biological species. The names T. yallundae and T. acuformis are accepted for the two species. Criteria to accurately distinguish the species are proposed based on mating ability and molecular markers, rather than less reliable tests comparing morphology in culture, and these are successfully used to differentiate a series of 30 test isolates.