The population structure of Armillaria gallica, an important pathogen of Quercus spp., was investigated from mixed oak forests in central Massachusetts, encompassing a sampling area over 500 km2. From 16 plots at four sites a total of 153 isolates (34–40 isolates per site) was analyzed with amplified fragment length polymorphisms (AFLPs). Analyses of 204 polymorphic loci detected 38 AFLP genotypes from a sample area of 4.51 hectares (ha). Genets ranged in distribution from five to 33 genets per hectare (GPH), with a mean of eight GPH and the average A. gallica genet occupying 0.13 ha. Allele frequencies produced an unbiased expected heterozygosity (HE) value of 0.112 (SE = 0.006) and a Nei’s expected heterozygosity (HJ) value of 0.190 (SE = 0.009), indicating low genetic diversity within the population. Analysis of molecular variation (ΦPT = 0.301; P < 0.001) indicates high genetic differentiation, with 70% of the molecular variation explained at the site-level within A. gallica subpopulations. However, results of the Mantel test, used to assess the isolation-by-distance hypothesis, were inconclusive in determining whether the subpopulations were truly isolated by distance. A neighbor-joining tree constructed from a genetic distance matrix grouped genotypes from the same site (subpopulation) together, but from three of four sites genotypes were randomly clustered at the plot level. The results suggest that basidiospore dispersal is an important means of new genet formation at linear distances up to 2000 m.