Roadkill is one of the highest causes of wildlife mortality and is of global conservation concern. Most roadkill studies have focused on wildlife in developed countries such as the United States of America and temperate biomes, but there are limited data for the impacts of roads on wildlife in the African tropics, where road infrastructure development is projected to grow rapidly in natural environments and conservation areas. The Tsavo Conservation Area is an important biodiversity hotspot in eastern Kenya and is bisected by a major highway and railways that connect the port of Mombasa to the interior. Along this infrastructure corridor, roadkill was recorded for 164 days over an 11-year period (2007–2018). In total, 1,436 roadkill were recorded from 13,008 km driven of a 164.42 km Nairobi-Mombasa road representing 0.11 collisions per kilometer. The majority of roadkill were small to medium sized mammals (<15kg) (53%; n = 756), whereas birds comprised 32% (n = 460), reptiles 10% (n = 143), with the remaining 5% (n = 77) being large mammals (>15kg). Of the 460 birds recorded, 264 were identifiable represented by 62 species. All large mammals comprising 10 species were identified, including the African elephant, Loxodonta africana and the endangered African wild dog, Lycaon pictus. Thirteen species of small mammal were also identified dominated by Kirk's dik-dik (Madoqua kirkii). Reptiles were represented by 11 species which were identified to the species level. Roadkill hotspots were identified using a kernel density method. The spatial distribution of roadkill was associated with adjacent shrub vegetation and proximity to permanent and seasonal rivers, and differences in seasonality and habitats were observed. Roadkill was lowest on road sections that traversed settled areas as opposed to roads adjacent to the protected areas. The results demonstrate that roadkill for two of the taxonomic groups - mammals and birds - appear high with numerous species detected in the Tsavo Conservation Area. These results can be used to focus efforts to reduce wildlife mortality by guiding future mitigation efforts.