Globally, traffic associated with roads that pass through protected areas are the cause of many negative impacts on wildlife, such as wildlife roadkill, which has occurred within the Serengeti ecosystem. A 200 km stretch of gravel road networks that passes through the Serengeti ecosystem was surveyed in 2015 for five consecutive days during each study period during the wet and dry seasons, between March and August 2015, to document the magnitude, patterns and composition of wildlife roadkill. We found a total of 51 wildlife roadkills that consisted of 26 species of wild animals and included 18 bird and 8 mammal species. Bird species (60.8%) were more frequently killed than mammal species (39.2%). Moreover, a higher incidence of wildlife roadkill was recorded in the morning (56.9%) than in the afternoon (43.1%) and more mammals (51.7%) than birds (48.3%) were recorded in the morning. Greater numbers of wildlife roadkill were recorded along road segments with good road conditions (69.4%) that were wider (>8 m) and with higher traffic volumes than along roads with poor conditions (30.6%). Overall, the Cape hare (Lepus capensis) was the most frequently killed species (17.6%) among the mammals, whereas the helmeted guineafowl (Numida meleagris) (13.7%) was most frequently killed among the avifauna. To reduce the number of roadkill incidents, we recommend changing driver speeding behaviours by installing wildlife-warning signage that consists of picture-based signs, because they have been proved to be more effective than word-based signs to reduce speeds and hence, reduce collisions. Additionally, the responsible management authorities should provide education to drivers on safe driving and enforcement of laws and regulations. Further, policy briefings that focus on wildlife roadkill should be made available to assist decision-makers and engineers to improve road design in protected areas.