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Microbial ethanol production is an important alternative energy resource to replace fossil fuels, but at high level, this product is highly toxic, which hampers its efficient production. Towards increasing ethanol-tolerance of Saccharomyces cerevisiae, the so far best industrial ethanol-producer, we evaluated an in vivo evolutionary engineering strategy based on batch selection under both constant (5%, v v⁻¹) and gradually increasing (5–11.4%, v v⁻¹) ethanol concentrations. Selection under increasing ethanol levels yielded evolved clones that could tolerate up to 12% (v v⁻¹) ethanol and had cross-resistance to other stresses. Quite surprisingly, diploidization of the yeast population took place already at 7% (v v⁻¹) ethanol level during evolutionary engineering, and this event was abolished by the loss of MKT1, a gene previously identified as being implicated in ethanol tolerance (Swinnen et al., Genome Res., 22, 975 …