Abstract Native to much of Central and South America, the little fire ant Wasmannia auropunctata has been rapidly spreading throughout the world. In its introduced range, W. auropunctata is frequently linked with drastic reductions of ant diversity; anecdotal reports of damaging attacks on vertebrates are also common. As it poses an ever-increasing threat to biodiversity, W. auropunctata has emerged as a model system for the study of ecological differences between native and invasive ant populations. These studies have been hampered by a lack of information on the genetic relatedness between native and introduced populations. By investigating the genetic structure of W. auropunctata populations, we provide a framework for conducting phylogenetically independent tests of differences between these ants in their native and invasive ranges. Phylogenetic analyses, based on the cox1/cox2 region of mtDNA, revealed at least three separate source populations of W. auropunctata distributed across two large clades. Much of the Caribbean region, presumably part of the native range, is inhabited by a clade of ants sharing very similar or identical mtDNA haplotypes, suggesting the possibility of multiple introductions or high levels of gene flow across that area. Most invasive populations in the Pacific were closely related to these ants. The invasive populations in Gabon and New Caledonia arise from another, relatively distantly related clade. Phylogenetically independent contrasts confirm McGlynn's (1999) observation that invasive W. auropunctata populations are smaller than native populations. Given the complex phylogeographical structure of W. auropunctata populations, future comparative work should correct for phylogenetic effects.