Short-tailed opossums (Monodelphis domestica) belong to the branch of marsupial mammals that diverged from eutherian mammals approximately 180 million years ago. They are small in size, lack a marsupial pouch, and may have retained more morphological characteristics of early marsupial neocortex than most other marsupials. In the present study, we used several different histochemical and immunochemical procedures to reveal the architectonic characteristics of cortical areas in short-tailed opossums. Subdivisions of cortex were identified in brain sections cut in the coronal, sagittal, horizontal or tangential planes and processed for a calcium-binding protein, parvalbumin (PV), neurofilament protein epitopes recognized by SMI-32, the vesicle glutamate transporter 2 (VGluT2), myelin, cytochrome oxidase (CO), and Nissl substance. These different procedures revealed similar boundaries among areas, suggesting that functionally relevant borders were detected. The results allowed a fuller description and more precise demarcation of previously identified sensory areas, and the delineation of additional subdivisions of cortex. Area 17 (V1) was especially prominent, with a densely populated layer 4, high myelination levels, and dark staining of PV and VGluT2 immunopositive terminations. These architectonic features were present, albeit less pronounced, in somatosensory and auditory cortex. The major findings support the conclusion that short-tailed opossums have fewer cortical areas and their neocortex is less distinctly laminated than most other mammals.