Transportation infrastructures such as roads, railroads and canals can have major environmental impacts. Ecological road effects include the destruction and fragmentation of habitat, the interruption of ecological processes and increased erosion and pollution. Growing concern about these ecological road effects has led to the emergence of a new scientific discipline called road ecology. The goal of road ecology is to provide planners with scientific advice on how to avoid, minimize or mitigate negative environmental impacts of transportation. In this review, we explore the potential of molecular genetics to contribute to road ecology. First, we summarize general findings from road ecology and review studies that investigate road effects using genetic data. These studies generally focus only on barrier effects of roads on local genetic diversity and structure and only use a fraction of available molecular approaches. Thus, we propose additional molecular applications that can be used to evaluate road effects across multiple scales and dimensions of the biodiversity hierarchy. Finally, we make recommendations for future research questions and study designs that would advance molecular road ecology. Our review demonstrates that molecular approaches can substantially contribute to road ecology research and that interdisciplinary, long-term collaborations will be particularly important for realizing the full potential of molecular road ecology.