Paved roads are a solution for communication between human societies, but at the same time, their expansion is detrimental to wildlife. In this work, vertebrate mortality events due to traffic collisions on National Route N° 5, in a 50 km stretch from the town of Pozo Colorado to the east, are evaluated. Vehicle journeys were carried out at a constant speed of 40 km/h every two months, from November 2020 to May 2022. All findings of roadkill were recorded by photograph. To analyse spatial patterns, the Kernel density was estimated, assessing the randomness of Ripley's K collisions and standardised roadkill rates were assessed for each species. A total of 272 individuals were recorded, corresponding to 87 amphibians, 38 birds and 35 mammals. In terms of the number of species, the composition was as follows: reptiles with 20 species, birds with 13 species, mammals with 11 and amphibians with at least 12 species. The species with the highest number of dead individuals was the common toad (Rhinella diptycha), followed by snakes. According to the standardised roadkill rates, the most affected animals are Cerdocyon thous, Rhinella diptycha, Caiman yacare and Dryophylax hypoconia with more than 200 individuals per kilometre per year. A bat, Lasiurus ega, was identified for the first time for Paraguay. Climatic conditions seems to have no strong effect on the occurrence pattern of the different taxa, with the exception of birds that decrease with stronger winds. The greatest coincidences occurred in three sections: km 5–5.5, km 33.5–34.5 and km 40–43. There was a correlation with crossroads areas, watercourses and forest islands. In view of the road development policy in the region, it is necessary to carry out studies of its impacts in the longer term.