The production of second-generation ethanol from hemicellulosic hydrolysate has received great attention lately due to its high economic interest. Mixture of molasses and hemicellulosic hydrolysates could be appropriate for sugar mills since they can provide all necessary nutrients for cells at low cost. However, naturally pentose-fermenting cells can hardly metabolize all sugars in this mixture. As an alternative to genetically modified Saccharomyces cerevisiae, this work investigated the improvement of naturally pentose fermenting wild yeasts, through evolutionary engineering. Firstly, Spathaspora passalidarum cells were sequentially cultivated in solid media composed by molasses and increasing concentrations of xylose. Finally, the evolved cells from the medium composed by molasses and xylose were inoculated in solid and liquid media composed by molasses, xylose and hemicellulosic hydrolysate. All evolved strains produced more ethanol than the parental strain (evolved strains SPF001 and SP024 showed a 33 and 55 % increase in the final ethanol production respectively). Strains SP F001 and SP025 produced YP/S of 0.42 and 0.41 g/g respectively, higher than that of the parental strain (0.34 g/g). Therefore, evolutionary engineering generated more efficient cells regarding sugar consumption and ethanol production.