To investigate the use of genome shuffling to generate recombinants from previously generated hydrolysates-tolerant strains to improve tolerance of Saccharomyces cerevisiae to one or more inhibitory by-products present in lignocellulosic hydrolysates. Recombinants of previously evolved strains of S. cerevisiae were generated and analyzed for their relative performance in the individual inhibitors furfural, acetic acid, 5-(hydroxymethyl)-furfural (HMF) and in synthetic hydrolysates. One recombinant exhibited a 100 % fitness increase in the presence of HMF as compared to the wild-type diploid, while another stain exhibited a 13 % fitness increase in the presence of furfural. Furthermore, for one of these recombinants, these increases in fitness were specific to the inhibitor HMF and to synthetic hydrolysates rather than being due to a general increase in fitness. Mutations present in the evolved hydrolysates-tolerant mutants were identified via whole-genome resequencing. Recombinants of S. cerevisiae were produced with increased tolerance to inhibitory by-products present in hydrolysates of lignocellulosic biomass and identified potential genetic determinants associated with this phenotype.