A fundamental tenet of sociobiological theory is that differences in reproductive ability within eusocial colonies should be environmentally, not genetically based. However, populations of seed harvesting ants in the Southwestern US ( Pogonomyrmex rugosus and P. barbatus ) have a genetic caste determination system, in which workers are heterozygous at multiple loci, while their reproductively capable sisters (alates) are homozygous. Our data show that this phenomenon is widespread across the ranges of overlap for the two species, supporting the hypothesis that it is linked in some way to hybridization events between the species. However, its persistence in areas outside hybrid zones suggests this is not a complete explanation for its evolution. Linkage mapping indicates that a large portion of the genome is involved in caste determination, rather than a single “caste gene”. Finally, caste determination is driven in part by colony behavior. Queens of colonies showing caste determination lay homozygous eggs that do not develop into workers, suggesting that homozygous females may be culled during brood care.