The army ant Eciton burchellii is probably the most important arthropod predator in the Neotropics, and many animal species depend upon it. Sex-biased dispersal with winged males and permanently wingless queens may render this species especially sensitive to habitat fragmentation and natural barriers, which might have severe impacts on population structure and lead to population decline. Using nuclear microsatellite markers and mitochondrial sequences, we investigated genetic differentiation in a fragmented population in the Panama Canal area. While nuclear markers showed little differentiation between subpopulations (FST = 0.017), mitochondrial differentiation was maximal in some cases (ΦST = 1). This suggests that, while females are not capable of crossing barriers such as large rivers, flying males are able to promote nuclear gene flow between the studied forest patches. Consistent with this interpretation, we did not find any evidence for inbreeding or genetic deterioration on Barro Colorado Island over the last 90 years since its formation.