We monitored reptile and bird roadkills in Cerrado–Pantanal landscapes along the Campo Grande to Corumbá highway BR-262. We describe species distribution in different landscape zones, including the first geographic record for Hydrodynastes bicinctus Herrmann, 1804 in the Pantanal basin. The roadkill occurrence of Spizaetus melanoleucus (Vieillot, 1816) is an outstanding record. We recorded 930 individuals belonging to 29 reptile and 47 bird species; 20 of these species are new roadkill records in Brazil. The 8 new records of reptile species include Eunectes notaeus Cope, 1862, Bothrops mattogrossensis Amaral,1925, Dracaena paraguayensis Amaral,1950 and H. bicinctus; and 12 new records of bird species include S. melanoleucus, Heterospizias meridionalis Latham, 1790, Urubitinga urubitinga (Gmelin, 1788), Pulsatrix perspicillata (Latham, 1790), Aramus guarauna (Linnaeus, 1766), and Jabiru mycteria (Lichtenstein, 1819). Richness of road-killed species on the BR-262 highway seemed to be high, reinforcing concerns about wildlife-vehicle collisions where these accidents occur, as they lead to long term and chronic impacts on wildlife and road safety in the Pantanal region.