Linear transportation infrastructure structures are detrimental to the existence of macro-ecosystems in two main ways, causing mortality of the organisms and imposing limitations on the spatial processes of top predators. This study explores the consequences of roadkill mortality and vehicular disturbance for genetic connectivity and behavioral adaptation to roads in apex predators. By employing a descriptive approach through landscape synthesis, the present work analyzes both the impact of roadkill mortality leading to a reduction in local populations and the influence of vehicular disturbance creating a behavioral barrier for the passage of apex predators across roads. The proposed methodology involves the combination of fieldwork observations, non-invasive genetic analyses, and GPS telemetry tracking in order to assess the effects of small-scale displacement processes and fragmentation of the genetic gene pool in highly mobile apex predators. It is found that despite the capacity of large carnivores to adjust their activity rhythms and cross linear barriers in periods of decreased traffic, extremely trafficked highways serve as complete obstacles for migration. Such a road impact leads to increased allelic isolation and inbreeding depression.