Symbiont choice has been proposed to play an important role in shaping many symbiotic relationships, including the fungus-growing ant-microbe mutualism. Over millions of years, fungus-growing ants have defended their fungus gardens from specialized parasites with antibiotics produced by an actinomycete bacterial mutualist (genus Pseudonocardia). Despite the potential of being infected by phylogenetically diverse strains of parasites, each ant colony maintains only a single Pseudonocardia symbiont strain, which is primarily vertically transmitted between colonies by the founding queens. In this study, we show that Acromyrmex leaf-cutter ants are able to differentiate between their native actinomycete strain and a variety of foreign strains isolated from sympatric and allopatric Acromyrmex species, in addition to strains originating from other fungus-growing ant genera. The recognition mechanism is sufficiently sensitive for the ants to discriminate between closely related symbiont strains. Our findings suggest that symbiont recognition may play a crucial role in the fungus-growing ant-bacterium mutualism, likely allowing the ants to retain ecological flexibility necessary for defending their garden from diverse parasites and, at the same time, resolve potential conflict that can arise from rearing competing symbiont strains.