Root system architecture (RSA) describes the dynamic spatial configuration of different types and ages of roots in a plant, which allows adaptation to different environments. Modifications in RSA enhance agronomic traits in crops and have been implicated in soil organic carbon content. Together, these fundamental properties of RSA contribute to the net carbon balance and overall sustainability of biofuels. In this article, we will review recent data supporting carbon sequestration by biofuel crops, highlight current progress in studying RSA, and discuss future opportunities for optimizing RSA for biofuel production and soil carbon sequestration.