Pathogenic fungi can have devastating effects on agriculture and health. One potential challenge in dealing with pathogens is the possibility of a host jump (i.e., when a pathogen infects a new host species). This can lead to the emergence of new diseases or complicate the management of existing threats. We studied host specificity by using a hybrid fungus formed by mating two closely related fungi: Ustilago bromivora, which normally infects Brachypodium spp., and U. hordei, which normally infects barley. Although U. hordei was unable to infect Brachypodium spp., the hybrid could. These hybrids also displayed the same mating-type bias that had been observed in U. bromivora and provide evidence of a dominant spore-killer-like system on the sex chromosome of U. bromivora. By analyzing the genomic composition of 109 hybrid strains, backcrossed with U. hordei over four generations, we identified three regions associated with infection on Brachypodium spp. and 75 potential virulence candidates. The most strongly associated region was located on chromosome 8, where seven genes encoding predicted secreted proteins were identified. The fact that we identified several regions relevant for pathogenicity on Brachypodium spp. but that none were essential suggests that host specificity, in the case of U. bromivora, is a multifactorial trait which can be achieved through different subsets of virulence factors.