Wolbachia are intracellular bacteria that induce phenotypic effects in many arthropod hosts to enhance their own transmission within host populations. Wolbachia commonly infect the Red Imported Fire Ant, Solenopsis invicta, in native South American populations. A previous study failed to detect Wolbachia in fire ants from the introduced range in the USA. We conducted an extensive study of individuals collected from 1157 nests from 10 widespread geographical populations in the USA. Wolbachia were detected in ants from two nearby populations in southern Mississippi, with different variants (wsp gene sequences) infecting ants from colonies of the multiple-queen (polygyne) vs. single-queen (monogyne) social forms. The parsimonious explanation for the presence of Wolbachia in introduced S. invicta is that there have been one or more recent introductions of Wolbachia-infected fire ants into the southern USA.