In order to differentiate the two genetic groups (A and B) detected in populations of the powdery mildew fungus, *Uncinula necator*, we developed PCR primers to amplify two sequence characterized amplified regions (SCAR) and one sequence with a microsatellite motif (SSR). Primer pairs designed from SCARs gave a fragment for group A isolates and no fragment for group B isolates. The primer pair mO3E11 designed for the SSR sequence amplified two different sequences. The first sequence had the SSR motif and presented two alleles: one allele was present in all 49 isolates of group A tested and in two out of 34 isolates of group B, and a second allele was present in the remaining 32 isolates of group B. The other sequence did not have the SSR motif and was amplified only in the 34 group B isolates. The mO3E11 pair appears suitable for large-scale studies since it identified groups in only one PCR reaction and gave strong signals with DNA templates that can be rapidly obtained from a minute quantity of fungal material.