Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based. on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.