The fast evolution of Plasmopara halstedii (downy mildew) remains a major risk for sunflower crop, as new races of the pathogen are bypassing the resistance of sunflower hybrids. In order to understand the processes which led a new virulence to appear in a local P. halstedii population, the genetic relationships were studied using 12 EST (Expressed Sequence Tag)-derived markers between five progeny isolates of races 300, 304, 314, 704 and 714 and two parental ones of races 100 and 710. All genetic analyses were carried out using five single zoosporangium isolates per P. halstedii isolate. There was no intra-isolate genetic variation among the seven pathogen isolates and five multilocus genotypes (MLG) were identified among the 35 P. halstedii single zoosporangium isolates. The single zoosporangium isolates of races 314, 704 and 714 had an intermediary genetic position between the single zoosporangium isolates of two parental isolates. The single zoosporangium isolates of three isolates of races 100, 300 and 304 were localized in the same genetic clade. Two genetic mechanisms could explain the emergence of new virulence in P. halstedii as a recombination between races and mutations in a clonal lineage.