In recent decades, rapid industrial growth has accelerated the construction of new roads, which has led to the destruction and isolation of wildlife habitats. Newly constructed roads affect wildlife in many ways. In particular, fatal wildlife-vehicle collisions (WVCs) have a direct impact on wildlife. A substantial number of WVCs occur every year on expressways, where vehicle speeds and vehicle traffic are significant. However, our understanding of the relative importance of the factors associated with areas in which large numbers of WVCs occur on the expressway remains poor. Therefore, herein, we analyze the spatial characteristics of WVCs. The effect of spatial distribution on the occurrence of WVCs was analyzed using the types of land cover in the areas where water deer appear (Cheongju, Boeun, and Sangju) and the areas in which WVCs occur along the Cheongju–Sangju Expressway (CSE). We identified the WVC hotspots by using CSE patrol data recorded between January 2008 and December 2019, and we analyzed the corresponding distribution patterns and land cover characteristics. Along the CSE, a total of 1082 WVCs occurred, out of which collisions involving water deer (Hydropotes inermis argyropus) accounted for 91%. Water deer appear frequently in Forested Areas and Agricultural Land, but the WVC distribution in the Hotspots followed a highly clustered pattern, with a higher proportion of WVCs occurring in Used Areas (areas including buildings such as residential facilities, commercial and industrial facilities, and transportation facilities). Used Areas have a smaller cut slope compared to Forested Areas, and Used Areas are open terrains. Therefore, the occurrence of WVCs will be high given that wildlife can easily access the expressway. Based on these results, we can infer that the landscapes near the expressway influence the occurrence of WVCs. To establish an effective policy for reducing WVCs on a road, the WVC characteristics and spatial distribution of the road should be considered together. Further research on the wildlife ecology and land-use status of WVC hotspots is required to mitigate WVCs on expressways and protect human and animal life. Therefore, if the characteristics of WVC hotspots are analyzed considering the characteristics of various ecosystems, an appropriate WVC reduction plan can be established.