Biofuel technologies continue to be researched and improved and biofuel policies are being debated and shaken up. Ultimately, success of large-scale biofuel deployment depends on biofuel economics in the marketplace and true biofuel sustainability in political and social context. This special section in Biotechnology for Biofuels presents a collection of articles in the areas of techno-economic analysis, sustainability analysis and life-cycle analysis of biofuels for road and aviation transportation applications. The articles cover a variety of biomass feedstocks, conversion technologies, and biofuel end uses along the whole biofuel supply chain. They address critical issues such as economic drivers for biofuel costs, greenhouse gas emissions, water use impacts, and indirect land use changes. The up-to-date research results on these topics from authors will help the biofuel community identify biofuel R&D opportunities and challenges and the policy community develop sound, consistent biofuel policies so that biofuels will indeed play an important role in pursuing long-term sustainability of the transportation sector globally.