Roads impact wildlife around the world; however, dedicated studies are lacking in many biodiverse areas such as the Amazon. Identifying which species are more often hit by vehicles and which landscape and road-related features promote roadkill is essential to guide future development and ensure adequate mitigation actions. For six months, we monitored 240 km of roads in the Ecuadorian Amazon and recorded 1125 dead vertebrates (149 species). Reptiles were the most observed Class with 380 individuals (56 species), followed by amphibians with 278 individuals (11 species), birds with 259 individuals (62 species), and mammals with 208 individuals (20 species). We used Random Forest models to explore the role of various land cover types and road sinuosity on the observed mortality. Additionally, we created heatmaps to visualize the road segments where roadkills were more frequent. For all vertebrates, mortality was more likely in straight road sections near rivers. The effects of other variables were taxa-specific. Amphibian mortality was more likely near bare soil or forest, birds and mammals died more often near herbaceous-shrubby vegetation, and reptile mortality occurred more often in areas with high cover of agriculture. Road segments with high mortality (i.e., roadkill hotspots) varied across taxa. These hotspots identify areas where further research into mitigation is needed to assess road impacts and prevent collisions. Among records, we found rare and threatened species, for which road mortality could be a significant threat. Roadkill surveys not only aid in quantifying threats and informing future planning but can also provide insight into local biodiversity.