Unicoloniality, or the absence of behavioral boundaries between nests, is thought to promote ant abundance due to reduced intraspecific competition. Workers within unicolonial populations may increase their own inclusive fitness by preferentially caring for more related individuals (nepotism), but nepotism has only rarely been documented in ants. We tested for unicoloniality and nepotism in polygyne red imported fire ants (Solenopsis invicta; hereafter fire ants). Fire ants occur in two social forms: monogyne (i.e., colonies with a single egg-laying queen) and polygyne (i.e., colonies with multiple egg-laying queens). Introduced populations of polygyne fire ants are commonly referred to as unicolonial, but cooperation between and within colonies is poorly documented. To delimit boundaries between colonies in the field, we quantified the exchange of a ¹⁵N-glycine tracer dissolved in a sucrose solution and correlated this exchange with colony genetic structure. We also quantified within-colony conflict between workers and larvae using close siblings (i.e., from the same mother) and non-siblings (i.e., from a different mother). Counter to our expectations, polygyne colonies did not exchange resources or workers, indicating distinct colony boundaries. Polygyne workers also preferentially fed larval sibling and may have preferentially cannibalized non-siblings. Polygyne colony behavior was correlated with higher levels of within-mound relatedness between workers in the field than those previously reported in North America (mean ± SE: 0.269 ± 0.037). Our study challenges fundamental assumptions about introduced populations of polygyne fire ants and suggests that polygyne colonies are multicolonial and likely engage in high levels of intraspecific competition.