Verticillium dahliae is widely distributed in potato and olive fields in Lebanon, causing serious economic losses. However, little is known about the inoculum source, population structure, and genetic diversity of the pathogen or the mechanisms of dissemination within Lebanon. To understand the population structure, a total of 203 isolates sampled from olive (n = 78) and potato (n = 125) were characterized for species, mating type, and race, and the genetic relationships were delineated using 13 microsatellite markers. All isolates except one from potato were V. dahliae, with 55.1 and 12.1% race 1, and 43.6 and 83.1% race 2 in olive and potato, respectively. The genetic structure of the studied population was best described by two large and two small clusters. Membership in the two large clusters was determined by the presence or absence of the effector gene Ave1. Furthermore, genetic structure was moderately associated with the host of origin but was weakly associated with the geographic origin. All but four isolates represented by three multilocus haploid genotypes were MAT1-2. This study identified a clear lack of gene flow between virulence genotypes of V. dahliae despite the proximity of these cropping systems and the wide distribution of genetic diversity among hosts and geographic regions in Lebanon.