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Abstract Second-generation bioethanol production using lignocellulosic biomass as feedstock requires a highly efficient multistress-tolerant yeast. This study aimed to develop a robust yeast strain of P. kudriavzevii via the adaptive laboratory evolution (ALE) technique. The parental strain of P. kudriavzevii was subjected to repetitive long-term cultivation in medium supplemented with a gradually increasing concentration of acetic acid, the major weak acid liberated during the lignocellulosic pretreatment process. Three evolved P. kudriavzevii strains, namely, PkAC-7, PkAC-8, and PkAC-9, obtained in this study exhibited significantly higher resistance toward multiple stressors, including heat, ethanol, osmotic stress, acetic acid, formic acid, furfural, 5-(hydroxymethyl) furfural (5-HMF), and vanillin. The fermentation efficiency of the evolved strains was also improved, yielding a higher ethanol concentration, productivity, and yield than the parental strain, using undetoxified sugarcane bagasse hydrolysate as feedstock. These findings provide evidence that ALE is a practical approach for increasing the multistress tolerance of P. kudriavzevii for stable and efficient second-generation bioethanol production from lignocellulosic biomass.