Acromyrmex leaf-cutting ants maintain two highly specialized, vertically transmitted mutualistic ectosymbionts: basidiomycete fungi that are cultivated for food in underground gardens and actinomycete Pseudonocardia bacteria that are reared on the cuticle to produce antibiotics that suppress the growth of Escovopsis parasites of the fungus garden. Mutualism stability has been hypothesized to benefit from genetic uniformity of symbionts, as multiple coexisting strains are expected to compete and, thus, reduce the benefit of the symbiosis. However, the Pseudonocardia symbionts are likely to be involved in Red-Queen-like antagonistic co-evolution with Escovopsis so that multiple strains per host might be favoured by selection provided the cost of competition between bacterial strains is low. We examined the genetic uniformity of the Pseudonocardia symbionts of two sympatric species of Acromyrmex ants by comparing partial sequences of the nuclear Elongation Factor-Tu gene. We find no genetic variation in Pseudonocardia symbionts among nest mate workers, neither in Acromyrmex octospinosus, where colonies are founded by a single queen, nor in Acromyrmex echinatior, where mixing of bacterial lineages might happen when unrelated queens cofound a colony. We further show that the two ant species maintain the same pool of Pseudonocardia symbionts, indicating that horizontal transmission occasionally occurs, and that this pool consists of two distinct clades of closely related Pseudonocardia strains. Our finding that individual colonies cultivate a single actinomycete strain is in agreement with predictions from evolutionary theory on host–symbiont conflict over symbiont mixing, but indicates that there may be constraints on the effectiveness of the bacterial symbionts on an evolutionary timescale.