Clonal isolates of Uncinula necator, the grapevine powdery mildew pathogen, were characterized by mating type, RFLP and PCR analyses. Five random genomic clones, consisting of three multi-copy and two low-copy sequences, were used as probes for hybridization. The oligonucleotides (CAC)5, (GACA)4, the core sequence of the M-13 minisatellite region and the consensus region from a plant intron splice junction (R1), were used as primers in PCR reactions. The DNA probes detected high levels of genetic diversity in U. necator, identifying 34 unique haplotypes among the 81 isolates analysed, with a Shannon diversity index of D′=0·85. In comparison, 15 haplotypes were resolved in PCR reactions with the four primers, resulting in a diversity index of D′=0·59. The overall diversity within the collection, by combining RFLP and PCR data, was D′=0·88, distinguishing 37 haplotypes. The RFLP analysis provided greater discrimination of individual haplotypes than did PCR. Phenetic analysis of the RFLP and PCR data separately and in combination revealed two broad phenetic groups. The average overall similarity between the two groups was 40%. Mating of isolates from the two phenetic groups resulted in viable ascospores. The combination of RFLP, PCR and mating techniques was effective in determining levels of diversity and provided insight into the genetic relatedness of U. necator isolates.