In polygynous (multiple queens per nest) colonies of social insects, queens can increase their reproductive share by laying more eggs or by increasing the proportion of eggs that develop into reproductive individuals instead of workers. We used polymorphic microsatellite loci to determine the genetically effective contribution of queens to the production of gynes (new queens), males, and 2 different cohorts of workers in a polygynous population of the ant Formica exsecta. For this purpose, we developed a new method that can be used for diploid and haplodiploid organisms to quantify the degree of reproductive specialization among breeders in societies where there are too many breeders to ascertain parentage. Using this method, we found a high degree of reproductive specialization among nest-mate queens in both female- and male-producing colonies (sex ratio is bimodally distributed in the study population). For example, a high effective proportion of queens (25% and 79%, respectively) were specialized in the production of males in female- and male-producing colonies. Our analyses further revealed that in female-producing colonies, significantly fewer queens contributed to gyne production than to worker production. Finally, we found significant changes in the identity of queens contributing to different cohorts of workers. Altogether, these data demonstrate that colonies of F. exsecta, and probably those of many other highly polygynous social insect species, are composed of reproductive individuals differing in their investment to gynes, males, and workers. These findings demonstrate a new aspect of the highly dynamic social organization of complex animal societies.