Nestmate recognition among social insects is presumed to restrict non-nestmates from exploiting nest resources. Here, we developed aggression bioassays to assess the discrimination behaviors of both polygynous and monogynous forms of the red fire ant, Solenopsis invicta Buren, during symmetrical interactions in neutral arenas. Workers from polygyne colonies exhibited risk avoidance behaviors; that is, defensive postures or the avoidance of direct contact during interactions. Workers from monogyne colonies always exhibited aggressive behaviors in the form of physical or chemical attacks. In interactions between both, monogyne workers usually started the aggression by surrounding and biting the polygyne ants. Polygyne S. invicta workers also distinguished nestmates from foreigners, but their response was not as aggressive as that of monogynes. The proposed ethogram that we constructed identified monogyne and polygyne forms of S. invicta colonies in concordance with current measures, including number of queens, and expression of the Gp-9 gene.