Abstract A comprehensive species-level phylogeny of the ant genus Linepithema Mayr, a Neotropical group best known for the invasive Argentine ant L. humile (Mayr), is inferred for the first time using fragments from three nuclear loci [wingless (WG), long-wavelength rhodopsin (LWR) and internal transcribed spacer (ITS-2)] and the mitochondrial cytochrome oxidase subunit I (COI) gene. Monophyly of the genus is strongly supported in parsimony, likelihood and Bayesian analyses of the concatenated data, as is the monophyly of four species groups defined previously on the basis of morphology. An Andean species, L. oblongum (Santschi), is the sister taxon of the Argentine ant. Eight of the 11 species whose monophyly was testable in the analysis were inferred to be monophyletic. Several instances of species paraphyly and one case of mitochondrial introgression suggest that complex population genetic processes underpin the patterns of diversity in Linepithema, and that simple genetic approaches to taxonomy such as DNA barcoding should be treated with caution. A maximum likelihood reconstruction of ancestral distributions suggests that Linepithema is of southern South American origin and that populations in the Greater Antilles are the result of four independent colonization events.