Technical Abstract: The ecological and evolutionary dynamics of newly introduced invasive species can best be understood by identifying the source population(s) from which they originated, as many species vary behaviorally, morphologically, and genetically across their native landscapes. We attempt to identify the source(s) of the red imported fire ant (Solenopsis invicta) in the southern U.S.A. utilizing data from three classes of genetic markers (allozymes, microsatellites, and mitochondrial DNA sequences) and employing Bayesian clustering simulations, assignment and exclusion tests, and phylogenetic and population genetic analyses. We conclude that the Mesopotamia flood plain near Formosa, Argentina represents the most probable source region for introduced S. invicta among the ten localities sampled across the native South American range. This result confirms previous suspicions that the source population resides in northern Argentina, while adding further doubts to earlier claims that the Pantanal region of Brazil is the source area. Several lines of evidence suggest that S. invicta in the southern U.S.A. is derived from a single source rather than the product of multiple invasions from widely separated source localities. Although finer-scale sampling of northern Argentina and Paraguay combined with the use of additional genetic markers will be necessary to provide a highly precise source population assignment, our current results are of immediate use in directing future sampling and focusing ongoing biological control efforts.