Molecular Road Ecology is a discipline that studies how road networks affect gene flow, reduce genetic diversity, and reshape population structure across fragmented landscapes. In this chapter, we review the existing literature demonstrating how landscape genetics provides a useful framework for assessing these impacts and can contribute to understanding and mitigating the genetic consequences of roads on biodiversity. We also present a brief overview of the most commonly used genetic sampling methods, molecular techniques, and analytical approaches applied in this discipline. In the second part, we identify key research needs, highlight emerging areas of inquiry, and outline promising directions for future work. These include expanding the spatial and temporal scope of future studies, incorporating underrepresented taxa, moving from single-species to community-level analyses, applying approaches such as environmental DNA, metabarcoding, genomics, and functional markers, shifting from pattern description to process-based understanding, adopting predictive modeling, standardizing and sharing data, integrating socio-ecological dimensions, and strengthening the science–policy interface. Tackling these challenges can refine the discipline, improve our understanding of how road networks affect biodiversity at the genetic level, and support more effective mitigation strategies.