Microcell fusion is a technology that involves the transfer of single or small clusters of intact chromosomes from one cell to another. The transferred chromosome can be stably retained in the recipient cell background under dominant selective pressure. Hybrid clones generated by this method result in karyotypically simple and homogeneous populations that are excellent resources for physical mapping. This article will describe a general strategy for the efficient micronucleation of human and rodent cell populations and their use as donors for microcell fusion into recipient cell lines.