Cephalotes atratus is a common and widespread arboreal ant with a Neotropical distribution. C. atratus is particularly interesting from a phylogeographic perspective because it has a wide distribution, ranging from Panama to northern Argentina. Central and South America have undergone a complex geologic and climatic history, and the phylogeographic patterns of this species in this region have likely be influenced by the formation of the Isthmus of Panama, uplift of the Andes, and habitat fluctuations during the Pleistocene. From September 2006 to September 2007 I collected C. atratus for molecular analysis, focusing on the northeastern part of its range. I sequenced a mitochondrial DNA gene (cytochrome c oxidase I) to reveal patterns of genetic differentiation in this species. Though sampling has been geographically limited, interesting patterns emerge, suggesting the possibility that C. atratus originated in South America and subsequently colonized Central America. Results of phylogenetic analyses are presented and compared to patterns in other taxa, the geological record, and geographic and habitat variation. This study is the first to employ molecular techniques to this diverse Neotropical ant genus.