Based on whole-genome sequences, strain reconstruction studies for improved industrial applications have been initiated. For the construction of a custom-designed microorganism, an efficient genetic engineering tool which can transfer large genomic segments carrying useful genomic information is needed. We developed a novel and efficient method called genome swapping using Cre/lox site-specific recombination and a γ-ori replication system that can transfer large genomic segments between various microorganisms. In this study, we attempted to construct an IS-free Escherichia coli B strain, which is a popular industrial host strain in the production of useful biomaterials. However, mobile DNA elements such as insertion sequence elements (IS) and transposases caused a genetic unstableness. To eliminate this problem, we targeted IS elements for removal in 5 target regions and deleted IS elements within these target regions. The genome swapping method may be used to transfer IS-deleted target regions of the E. coli chromosome in one cell for the rapid construction of an IS-free E. coli B strain. Also, it is possible to construct a minimized genome and a hybrid microorganism for use in bioindustry and biotechnology.