The Landes de Gascogne (LdG) forest (south-western France) is a mostly artificial maritime pine (Pinus pinaster) monoculture of about one million hectares surface, which is intensively managed for timber and pulp production. Armillaria ostoyae is the most important fungal pathogen in this forest; its maximal incidence is observed in the coastal area but an increasing presence of the pathogen is reported on the interior. In the present study, we analyzed the genetic diversity of A. ostoyae in the LdG forest, with particular emphasis on the geographic differentiation and colonization history. A total of 541 A. ostoyae samples representing 28 different populations were screened at five microsatellite loci. The overall A. ostoyae population was composed of 98 genotypes and no genotype was shared among populations. The genetic diversity significantly decreased from the coastal area toward the interior, which suggests a colonization of the forest from the coast. The absence of correlation between genetic and geographical distances among populations indicates that colonization has probably occurred following a randomly pattern. Geographic groups of populations were lowly differentiated and genotypes were not more closely related within than among populations, suggesting important gene flow among populations.