Ecuador has both high richness and high endemism of species which are increasingly threatened by anthropic pressures, including roads. However, research evaluating the effects of roads remains scarce, making it difficult to develop mitigation plans. Here we present the first national assessment of wildlife mortality that allow us to 1) identify species and areas where mortality occurs due to collision with vehicles and 2) reveal knowledge gaps. We bring together data from systematic surveys and citizen science efforts in Ecuador to present a dataset with >5000 wildlife roadkill records from 454 species. Systematic surveys were reported by ten studies conducted in five out of the 24 Ecuadorian provinces. Collectively they revealed 282 species with mortality rates ranging from 0.008 to 95.56 ind./km/year. The highest rates were for the yellow warbler Setophaga petechia in Galápagos (95.56 ind./km/year), the cane toad Rhinella marina in Napo (16.91 ind./km/year), and the small ground-finch Geospiza fuliginosa in Galápagos (14.11 ind./km/year). Citizen science and other no systematic monitoring provided 1705 roadkill records representing all the 24 provinces of Ecuador and 299 species. The common opossum Didelphis marsupialis, the Andean white-eared opossum Didelphis pernigra, and the yellow warbler Setophaga petechia were more commonly reported (250, 104, and 81 individuals respectively). Across all sources, we found 15 species listed as Threatened and six as Data Deficient by the IUCN. We suggest stronger research efforts on areas where mortality of endemic or threatened species could be critical for populations, such as in Galápagos. This first assessment of wildlife mortality on Ecuadorian roads represents contributions from several sectors including academia, members of the public, and government underlining the value of wider engagement and collaboration. We hope these findings and the compiled dataset will guide sustainable planning of infrastructure in Ecuador and ultimately, contribute to reduce wildlife mortality on roads.