Commercial production of bioethanol from lignocellulosic hydrolysates requires efficient fermenting strains. The abilities of the strain to converting all types of sugars in the hydrolysate to ethanol in high yield and to effectively tolerating/metabolizing inhibitors are necessary. Genome shuffling is a novel method for breeding, and it has been applied in pharmaceutical and food industry. This review summarized the technique of genome shuffling including principle, process, applications and its prospect for strains improvement in ethanol production from lignocellulosic hydrolysates.