Acetic acid existing in a culture medium is one of the most limiting constraints in yeast growth and viability during ethanol fermentation. To improve acetic acid tolerance in Saccharomyces cerevisiae strains, a drug resistance marker-aided genome shuffling approach with higher screen efficiency of shuffled mutants was developed in this work. Through two rounds of genome shuffling of ultraviolet mutants derived from the original strain 308, we obtained a shuffled strain YZ2, which shows significantly faster growth and higher cell viability under acetic acid stress. Ethanol production of YZ2 (within 60 h) was 21.6% higher than that of 308 when 0.5% (v/v) acetic acid was added to fermentation medium. Membrane integrity, higher in vivo activity of the H+-ATPase, and lower oxidative damage after acetic acid treatment are the possible reasons for the acetic acid-tolerance phenotype of YZ2. These results indicated that this novel genome shuffling approach is powerful to rapidly improve the complex traits of industrial yeast strains.