The interaction between animal movement and roads is pervasive, but little is known of the effects of the land-use patterns in roadside landscapes on roadkill events. Here, we compared wildlife roadkill along two road stretches that cross landscapes with different land-use patterns, including the presence of protected areas in Central Brazil. Sampling was conducted in 2017 and 2018 in two seasons (dry and rainy). We expected roadkill events to be more frequent bordering the protected area. Roadkill occurred more frequently in the rainy season in the unprotected landscape. Birds were most frequently recorded in the unprotected (44%, n = 76) than in the protected landscape (37%, n = 48). The least recorded group in the unprotected landscape was Squamata (11%, n = 18), while mammals were less detected in the protected landscape (14%, n = 18). Classes 'agriculture' and 'savanna' were related to amphibian roadkill numbers. For Squamata, we observed the effect of the presence of forests in the protected landscape. Bird roadkill was affected by protection level, while the presence of pasture and the level of protection explained mammal roadkill. Differences in roadkill patterns reinforce the need for long-term management of this source of mortality for the Cerrado fauna.