Obligate mutualistic symbioses rely on mechanisms that secure host-symbiont commitments to maximize host benefits and prevent symbiont cheating. Previous studies showed that somatic incompatibilities correlate with neutral-marker-based genetic distances between fungal symbionts of Panamanian leaf-cutting ants, but the extent to which this relationship applies more generally remained unclear. Here we showed that genetic distances accurately predicted somatic incompatibility for symbionts irrespective of whether neutral microsatellites or AFLP markers were used, but that such correlations were weaker or absent in sympatric colonies. Further analysis showed that the symbiont clades maintained by and were likely to represent separate gene pools, so that neutral markers were unlikely to be similarly correlated with incompatibility loci that have experienced different selection regimes. We suggest that evolutionarily derived claustral colony founding by queens may have removed selection for strong incompatibility in fungi, as this condition makes the likelihood of symbiont swaps much lower than in , where incipient nests stay open because queens have to forage until the first workers emerge.