Fungus-growing ants, their cultivated fungi and the cultivar-attacking parasite Escovopsis coevolve as a complex community. Higher-level phylogenetic congruence of the symbionts suggests specialized long-term associations of host-parasite clades but reveals little about parasite specificity at finer scales of species-species and genotype-genotype interactions. By coupling sequence and amplified fragment length polymorphism genotyping analyses with experimental evidence, we examine (i) the host specificity of Escovopsis strains infecting colonies of three closely related ant species in the genus Cyphomyrmex, and (ii) potential mechanisms constraining the Escovopsis host range. Incongruence of cultivar and ant relationships across the three focal Cyphomyrmex spp. allows us to test whether Escovopsis strains track their cultivar or the ant hosts. Phylogenetic analyses demonstrate that the Escovopsis phylogeny matches the cultivar phylogeny but not the ant phylogeny, indicating that the parasites are cultivar specific. Cross-infection experiments establish that ant gardens can be infected by parasite strains with which they are not typically associated in the field, but that infection is more likely when gardens are inoculated with their typical parasite strains. Thus, Escovopsis specialization is shaped by the parasite's ability to overcome only a narrow range of garden-specific defences, but specialization is probably additionally constrained by ecological factors, including the other symbionts (i.e. ants and their antibiotic-producing bacteria) within the coevolved fungus-growing ant symbiosis.