Spatial isolation and geological history are important factors in the diversification and population differentiation of species. Here we describe distributional patterns of ants in the genus Acropyga across Papua New Guinea (PNG), a highly biodiverse but little-studied region. We estimate phylogenetic relationships among currently recognised species of Acropyga and assess population genetic structure of the widespread species, A. acutiventris, across lowland areas of the island. We find that species of Acropyga present in PNG diversified during the Pliocene, between six and two million years ago. Most species now exhibit a patchy distribution that does not show a strong signal of geological history. However, the population genetic structure of the widespread species A. acutiventris has been influenced by geography, habitat association and, possibly, historical habitat fragmentation. There is a significant effect of isolation-by-distance within continuous lowland forest, and proximity to Australia has had a larger impact in structuring populations of A. acutiventris in PNG than has the Central Papuan Cordillera. This study is the first to describe population genetic patterns of an ant species in Papua New Guinea.