ABSTRACT The recent breakdown of Vf, a major resistance gene to apple scab, provided an opportunity to analyze a population genetic process within the matching virulent subpopulation of the fungus Venturia inaequalis. We utilized the amplified fragment length polymorphism technique and allelic variation at four microsatellite loci to assess genetic structure of 133 isolates of V. inaequalis from a single commercial apple orchard sampled from one cultivar carrying the Vf gene (Judeline) and three cultivars devoid of the Vf gene. Both analyses indicated a strong decrease of the genetic diversity among isolates from the Vf cultivar compared with the high level of diversity among isolates from the three other cultivars. This leads to a high genetic differentiation between virVf and avrVf groups (F(ST) > 0.17). Analyses of the genetic distance between AFLP patterns based on the Jaccard index indicate that all virVf isolates could be assigned to a single clonal lineage. These results lead us to conclude that the clonal structure of the population isolated from the Vf cultivar is an example of a founder effect in response to a resistance gene breakdown and it is likely that this event occurred in the orchard during the sampling year.