Using high sensitive polymerase chain reaction (PCR), we previously demonstrated that selective elimination of sperm mitochondrial DNA occurred during early embryogenesis in mouse. To analyze the process morphologically in more detail, a non-invasive, real-time observation of sperm mitochondria was used. Transgenic mice that express green fluorescent protein (GFP) exclusively in mitochondria (mtGFP-tg mice) were generated. The fluorescence in mtGFP-tg mice was strong and stable enough to carry out repeated observations under confocal laser scanning microscopy. In these mtGFP-tg mice it was revealed that the sperm mitochondria were selectively eliminated from egg cytoplasm during the two-cell stage of early embryogenesis. Therefore, mtGFP-tg mice should contribute to studies on sequential or repeated analysis of mitochondria.