The interest in developing biofuels has rapidly increased during the last decades followed by a strong controversy about their sustainability. Diverting a large amount of land from agriculture to fuels, impacting forests and grasslands, loss of biodiversity due to large monocropped fields are some threats that inhibit the momentum towards a significant substitution of fossil fuels by biofuels. From a methodological point of view, several estimations of the reduction of greenhouse gas (GHG) emissions from biofuels lead to a large variability of results even if they address the same biofuel pathway. It has been shown that the methods used and the assumptions on data inventories, system boundaries, allocation of resources and emissions may significantly impact the results. In different countries and regions in the world, sustainability standards are being developed in order to limit the promotion of biofuels to those that are environmentally sound, socially responsible and economically effective.