The aims of this work were to obtain, by evolutionary engineering, an industrial strain of Saccharomyces cerevisiae tolerant to high concentrations of HMF and to determine the expression levels of genes previously described as responsible for this tolerance. Cells were grown under anaerobic and oxygen limited conditions, in the presence of glucose or sucrose as carbon sources. P6H9 strain presented high expression levels for genes ADH7 and ARI1 in presence of HMF. This tolerant strain also showed higher ethanol productivity, biomass formation and alcohol dehydrogenase activity comparing to sensitive strains. Results suggest that S. cerevisiae P6H9 strain presents potential to be used for second-generation ethanol production. (C) 2013 Elsevier Ltd. All rights reserved.