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Biofuels are predicted to constitute a large proportion of fuel consumption in the global transport sector under stringent climate mitigation polices in future. In Brazil, sugarcane ethanol has been widely deployed at large scale and an oriented expansion of sugarcane is considered to be a key measure to ensure the expected positive outcomes from biofuels. In this study, more recent and accurate georeferenced information were combined with an enhanced land use classification system to update the original Sugarcane Agroecological Zoning (SAEZ). We also excluded environmentally relevant areas (ERA) in our updated SAEZ to create a new set of reference maps for supporting future prioritization of areas for sugarcane ethanol expansion. We mapped 41.7 Mha as suitable land for sugarcane expansion in center-south region of Brazil, differently from the 50 Mha indicated in the original SAEZ. After excluding the ERA, 33.7 Mha was found to be suitable for sugarcane expansion, of which 20 Mha is currently occupied with pasture lands. About half of these areas were located in Mato Grosso do Sul and Minas Gerais states and an exploratory assessment of two microregions from these states demonstrated the importance of conducting regional assessments to prioritize the areas for a more sustainable sugarcane expansion to meet future bioenergy demands. • Our analysis identified an area 17% smaller than original agro-ecological zoning suitable for sugarcane expansion. • By excluding environmentally relevant areas, the total suitable area for sugarcane expansion is reduced to 33.7 Mha. • About two thirds of that area is currently occupied with pasture lands. • About half of suitable areas occupied with pasture lands are in two Brazilian states. • Local conditions are fundamental for oriented expansion of bioenergy production. [ABSTRACT FROM AUTHOR]