Segregation and recombination in sexual progeny of Phaeosphaeria spp. were analysed using three PCR based techniques which targeted arbitrary DNA sequence (random amplified polymorphic DNA, RAPD–PCR), microsatellites (microsatellite primed PCR, MP–PCR) and dispersed repetitive DNA elements (rep-PCR). Polymorphic DNA markers were not detected in three sets of P. avenae f. sp. triticea ascospores isolated sequentially from single asci. It is hypothesised that ascospores of P. avenae f. sp. triticea were produced from a sexual process in which fusion of genetically identical nuclei took place. On the other hand, polymorphic DNA fragments were found in three sets of eight P. nodorum isolates recovered from ascospores sequentially liberated from single asci produced by in vitro mated ‘+’ and ‘−’ strains. Among 172 polymorphic markers identified in two sets of isolates (ascospores) of the fungus, 171 segregated in a ratio 1[ratio ]1. We have demonstrated for the first time that the inheritance of PCR based markers in P. nodorum progeny follows Mendelian genetics. Thereby, a molecular proof for bipolar heterothallism of the fungus was provided. In contrast, of 48 polymorphic markers detected in the third cross, only 26 segregated in an expected Mendelian frequency 4[ratio ]4 and the other 22 demonstrated either aberrant segregation pattern or possessed new DNA fragments amplified in PCR which were not detected previously in parental DNA profiles. The reasons underlying the high frequency PMS type markers identified almost exclusively in one cross have yet to be conclusively determined. If such recombinants were found in the progeny of in vitro mated P. nodorum isolates it is obvious that they also occur under natural conditions. Thus, the study provides a molecular evidence for generation of novel genotypes of the pathogen.