Workers of the neotropical army ant species Eciton burchelli show a strong and spectacular worker caste polymorphism, where worker castes do not only differ in their size but also in their proportions and from of their mandibles. Moreover this strong polymorphism is accompanied with a distinctive polyethism of the worker castes. Since E. burchelli queens are highly polyandrous the colonies headed by them are genetically complex structures constituted of many subfamilies (Denny et al. 2004. 91:396–399). We analyzed the worker offspring of three E. burchelli colonies using eight highly variable microsatellites to determine the patriline composition in these colonies. We found that the patrilines were not equally distributed among the castes in the three E. burchelli colonies, but instead many patrilines showed a significant accumulation in specific worker castes. Based on these data we can therefore conclude that in E. burchelli caste determination has a strong genetic basis and polyandry is essential to establish efficient division of labour in E. burchelli colonies.