Genome shuffling is a process to combine advantage traits by the recombination of the entire genome and it has been successfully applied in the laboratory evolution of various industrial microorganisms. However, genome shuffling has not been described in Kluyveromyces marxianus (KM), a promising yeast host for the expression of heterologous proteins. In this protocol, genome shuffling in KM is performed by sexual reproduction and is combined with high-throughput screening to obtain high-yielding strains. Notably, the screening of diploid clones risen from one mating mixture is carried out to improve the effectiveness of evolution. Mating-sporulation-mating cycles are repeated to obtain KM strain with ideal traits. •The method combines genome shuffling with high-throughput to achieve strains displaying high yielding of heterologous proteins.•This method can be applied to the genome shuffling of other species when only a few starting strains are available for sexual reproduction.