A method of deleting a target region for deletion in a host DNA using a donor DNA: wherein the donor DNA comprising regions homologous to a 5'-side region outside of the target region for deletion in the host DNA, a 3'-side region outside of the target region for deletion in the host DNA and a first homologous recombination region inside of the target region for deletion in the host DNA, respectively, in this order, and further comprising a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3'-side region and the region homologous to the first homologous recombination region; which method comprises the steps of: a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5'-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3'-side region derived from the host DNA and the 3'-side region derived from the donor DNA, to conduct selection of a host whose target region is deleted based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter, wherein said selectable marker genes do not remain in said host DNA after deletion. wherein the donor DNA comprising regions homologous to a 5'-side region outside of the target region for deletion in the host DNA, a 3'-side region outside of the target region for deletion in the host DNA and a first homologous recombination region inside of the target region for deletion in the host DNA, respectively, in this order, and further comprising a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3'-side region and the region homologous to the first homologous recombination region; which method comprises the steps of: a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5'-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3'-side region derived from the host DNA and the 3'-side region derived from the donor DNA, to conduct selection of a host whose target region is deleted based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter, wherein said selectable marker genes do not remain in said host DNA after deletion. a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5'-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3'-side region derived from the host DNA and the 3'-side region derived from the donor DNA, to conduct selection of a host whose target region is deleted based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter, wherein said selectable marker genes do not remain in said host DNA after deletion.