The genetic variation between island and mainland populations of the leaf-cutter ant Atta cephalotes have been poorly studied, despite its importance to understand their local migration, regional structure, and the distribution of one of the most abundant species in the neotropics. Objective: To analyze the genetic variation of an insular population of this leaf-cutting ant in a larger regional context in Central and South America. Methods: The Cytochrome Oxidase I gene of Atta cephalotes was sequenced for the Gorgona Island and mainland populations in order to conduct an analysis of molecular variance, geneflow model selection, and to calculate divergence time of haplogroups circulating in the island. An individual assignment test was conducted to estimate the spatial genetic connectivity and the distribution for this ant in the Eastern Tropical Pacific. Results: a total of 33 samples from the island and the Colombian mainland were compared to 389 Genebank accessions of Atta cephalotes from Central and South America. The leaf-cutter ant in the Gorgona Island is closely related to the Colombian mainland, despite that the separation between the island and the mainland likely constitute a barrier to geneflow. The most common haplogroups in the mainland likely arrived at the island sometime late in the Pleistocene. However, anthropogenic influence in the recent colonization of the island by this leaf-cutting ant cannot be excluded. A map of connectivity shows that this population has geographic limits that largely overlap with a biogeographic region within the Eastern Tropical Pacific. This trans-andean population accumulates 23% of the maternally inherited diversity of the Atta cephalotes. Conclusions: The leaf-cutter ant in the Gorgona Island and the Colombian Pacific is closely related to the Central American population and the whole region harbor a significant segment of Atta cephalotes genetic diversity in South America.