Technical Abstract: Twenty-two microsatellite loci were characterized in the soilborne plant pathogenic fungus Verticillium dahliae by analysis of the genome sequence. All loci were polymorphic in at least two of three populations of V. dahliae tested and collected from lettuce, spinach and tomato. These loci were useful in genotyping isolates and highlighting differences in genetic diversity among the three tested populations. With this set of highly polymorphic markers, the prospective of migration, structure and evolutionary analyses is expected to provide insight into gene flow on seed and devise new strategies for development and release of resistant germplasm.