PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes. ; Fundação para a Ciência e a Tecnologia, Portugal, through: Grant SFRH/BDP/63831/2009 to Grant SFRH/BPD/77995/2011; Strategic Project PEst-OE/EQB/LA0023/2013; and Project BioInd NORTE-07-0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER.