Roads have many impacts on biodiversity, and the number of studies in Road Ecology in the past 20 years has increased considerably. However, studies focusing on the population level are still scarce in this field of research. Here, we present examples on how the use of population perspectives can improve the quality of results and mitigation proposals. For instance, the inclusion of roadkill information in population viability analyses may draw attention on the risk of extinction of wildlife populations, and the use of individual-based population modeling allows more accurate estimation of the effect of roads on habitat-carrying capacity. Researchers should strive to go beyond collecting roadkill, targeting to assess whether roads and other linear infrastructure affect population viability of species inhabiting road vicinity areas. Similarly, rather than limiting data collection to monitor passage use (road crossings), road ecologists should aim to know whether functional connectivity—at the population level—is compromised by roads, or if it was improved after road mitigation. This can be achieved, for instance, by using molecular approaches, ecological modeling, and/or designs in which population trends are studied before and after the construction of a road and compared with control sites (BACI designs). Unless we focus on population levels, we will not fully know how roads are affecting wildlife population persistence.