Two distinct forms of colony social organization occur in the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae): 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. This major difference in social organization is associated with genetic variation at a single gene (Gp-9) whereby all polygyne queens possess at least one b-like allele, while monogyne queens lack such b-like alleles and instead harbor B-like alleles only. Further, a recent study of native populations 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. TaqMan allelic discrimination assays were developed to survey the variable nucleotide sites associated with these three critical amino acid residues. The assays were validated by surveying nests of known social form from the species' introduced in the USA and from native South American ranges, as well as by comparing the results to Gp-9 sequence data from a subset of samples. The results demonstrate these new molecular assays consistently and accurately identify the variable nucleotides at all three sites characteristic of the B-like and b-like Gp-9 allele classes, allowing for accurate determination of colony social form.