The PCR based random amplified polymorphic DNA (RAPD) technique was used to examine the degree of genetic variation within an asexual species of symbiotic fungus. The fungus associated with the leaf-cutting ant species Atta cephalotes, was collected from nests in two distinct geographical regions (two isolates from Panama and five isolates from Trinidad). Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. As expected, isolates collected from different geographic regions showed the greatest genetic variation (less than half of the bands were shared in their RAPD profiles). However, our results also demonstrate that considerable genetic diversity exists amongst isolates of the asexual symbiotic fungus even within a small geographic region. This discernable variation suggests that RAPD markers may be useful for providing a basis for quantitative assessment of genetic relationships within the fungal symbionts of leaf-cutting ants.