During the course of a study into the tooth development of marsupials, an interesting feature concerning cusp development was revealed by micro-focus computed tomography analysis and alizarin red S staining of the dentition from 9 to 25 days after birth (as a pouch-young specimen) of the gray short-tailed opossum (Monodelphis domestica). The metacone was the first cusp to calcify on the upper first molar, which commenced calcification 13 days after birth. The paracone and protocone were the second and the third cusps to calcify respectively, followed by the external stylar cusps. This order of cusp calcification differed from the order on the upper deciduous molar and permanent molars of Didelphis virginiana belonging to the same family, in which the stylar cusps calcified earlier than the protocone.