The ants of the genus Rhopalothrix are diverse in the Neotropical region, with 14 of the 16 described species. Based on museum material and recent fieldwork, Rhopalothrix ants in Colombia were reviewed. Morphological analysis of the workers allowed delimitation of six species, including two new species, Rhopalothrix mandibularis Guerrero & Grajales, sp. nov. and Rhopalothrix mariaemirae Tocora, Fiorentino & Fernández, sp. nov. A new combination Rhopalothrix amati comb. nov. is proposed for Eurhopalothrix amati . A worker-based taxonomic key, high-definition images of the workers, and a distribution map of all Rhopalothrix species present in Colombia are provided.