The development of modern society depends on the connectivity of different regions, and in particular, the establishment of roads and highways. While roads may be fundamental to development, they tend to have negative impacts on the biodiversity of a region. The present study verifies the roadkill patterns of the crab-eating fox, Cerdocyon thous, in the context of the characteristics of the surrounding matrix. Specimens were collected from four highways in the Brazilian states of Pernambuco and Alagoas, between August 2019 and December 2020. The date, time, and location of each specimen was recorded, and the carcasses were removed from the highway to avoid replicating records. A total of 101 specimens were collected, with a 1:1 sex ratio. Roadkill hotspots were observed in environments to which C. thous is adapted, in particular open fields and the margins of areas with denser vegetation. Rainfall may influence the observed roadkill patterns, given that the hotspots coincided with areas that have annual rainfall of between 600 mm and 700 mm. The molecular analyses indicated that the sample collected encompasses individuals from two distinct genetic groups, which are both distributed throughout the study area, reflecting its reduced genetic diversity. This scenario is influenced by factors such as the pattern of anthropogenic impacts within the study area and the ecological characteristics of the species. The impacts of the roadkill hotspots can be mitigated by measures such as signposting during recruitment periods.