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Vertebrate road kills are a generalized problem around the world, but they are scarcely documented on Colombian highways, especially in peri-urban areas. This study describes the highway mortality of mammals in a six year-period (2008-2013) on Vía del Escobero (Envigado, Antioquia, Colombia). Mammal groups that presented the highest mortality rates were marsupials (54.3%), carnivores (25.7%), and rodents (17.5%). Although there was a lower diversity and fewer individual fatalities than in previous years for the same highway, this study reports a greater number of fatalities for some endangered and unknown species such as Leopardus tigrinus, Puma yagouaroundi, and Bassaricyon neblina. This study also added three new species to the road kill list on this highway.