Caste differentiation and reproductive division of labor are the hallmarks of insect societies. In ants and other social Hymenoptera, development of female larvae into queens or workers generally results from environmentally induced differences in gene expression. However, several cases in which certain gene combinations may determine reproductive status have been described in bees and ants. We investigated experimentally whether genotype directly influences caste determination in two populations of Pogonomyrmex harvester ants in which genotype-caste associations have been observed. Each population contains two genetic lineages. Queens are polyandrous and mate with males of both lineages , but in mature colonies, over 95% of daughter queens have a pure-lineage genome, whereas all workers are of F1 interlineage ancestry. We found that this pattern is maintained throughout the colony life cycle, even when only a single caste is being produced. Through controlled crosses, we demonstrate that pure-lineage eggs fail to develop into workers even when interlineage brood are not present. Thus, environmental caste determination in these individuals appears to have been lost in favor of a hardwired genetic mechanism. Our results reveal that genetic control of reproductive fate can persist without loss of the eusocial caste structure.