Technical Abstract: Two distinct forms of colony social organization occur in the fire ant Solenopsis invicta: Colonies of the monogyne social form are headed by a single egg-laying queen, whereas those of the polygyne social form contain multiple egg-laying queens. Recent studies have demonstrated that genetic variation at a single gene (Gp-9) is responsible for this major difference in social organization whereby all polygyne queens possess at least one b-like allele whereas monogyne queens lack such b-like alleles and instead harbor B-like alleles only. Extensive sequencing revealed that all b-like alleles in polygyne queens consistently contain three diagnostic amino acid residues: possession of only one or two of these critical residues is not sufficient for polygyny. We developed TaqMan assays to survey these critical sites and then validated our assays by surveying nests of known social form from the species¿ native South American range as well as by comparing our results to Gp-9 sequence data from a subset of samples. Unlike previous assays, we show our method is sensitive, reliable, rapid, and accurate for determining colony social form, which is critical for planned and future studies aimed at better understanding the evolutionary genetics of social behavior in fire ants.