Thirty-one clonal isolates of U. necator were taken from diseased grapes from three different vineyards in France and Germany. Samples were collected in April from typical ‘flagshoot’ symptoms, and in July and August from the same plants. The mating-type and the sensitivity to the fungicide triadimenol were determined for all isolates. Techniques for obtaining and characterizing isolates are described. Two isolates in the first sampling and eight in the second were resistant to triadimenol, with resistance factors ranging from 3·4 to 11·8. All isolates in the first sampling and nine out of the 21 isolates in the second were mating-type (+), the remaining isolates were mating-type (−). Genetic variation between all isolates was assessed using the RAPD technique. Phenetic analysis based on the 364 RAPD fragments obtained revealed two very distinct groups, one group containing nine out of the 10 isolates from the first sampling, the second group containing all the remaining isolates. Isolates clustered in the first group displayed 58 RAPD fragments specific to them. These isolates did not exhibit 60 RAPD fragments present in all other isolates. Molecular and biological data suggested that isolates clustering in the two groups represent two different biotypes of U. necator, which are likely to be genetically isolated.