INTRODUCTION: In mammalian species, the neocortex contains at least six histologically‑distinct layers. The main difference in the structural organization of marsupial and eutherian brains appears in interhemispheric connections. In marsupials, the biggest cerebral axonal bundle is the anterior commissure, while eutherians form an additional commissure called the corpus callosum. AIM(S): The aim of this work was to study and compare development of the neocortex and the formation of interhemispheric connections in the opossum and the mouse. METHOD(S): The time sequence of the formation of neocortical layers was examined using immunofluorescent staining with neuronal markers for deep layers (Tle4 and Ctip2) and upper layers (Cux1, Satb2 and Brn2). To study cortico‑cortical connections, anterograde dyes, DiA and DiI were used. Generation of deeper cortical layers started at postnatal day (P)3 and continued till P8, while upper layers were generated from P9 to P19. At P9, Tle4 and Ctip2 positive neurons were observed in deep neocortical layers. Starting from P12 to P14, many cortical neurons located on the migration route between the germinal zone and upper layers were stained with Cux1, Brn2, and Satb2. At P17, Cux1 and Satb2 labeled cells reached upper layers. The pattern and proportion of Satb2 and Ctip2 positive cells in the opossum neocortex at P19 were similar to that observed in the mouse cortex at embryonic day 18. For labeling axons, DiA and DiI were injected into cortical deep layers and upper layers, respectively. RESULTS: We found that DiA labeled axons reached the subcortical structures, while DiI labeled axons made connections between different neocortical areas. CONCLUSIONS: The pattern of development of neocortical layers in opossums and mice is similar. However, it is shifted in time. Neurogenesis of the cerebral cortex in opossums takes place after birth while formation of the neocortex in mice occurs during the embryonic period. FINANCIAL SUPPORT: This work was supported by grant no. 2016/22/M/NZ4/00670 from the National Science Center Poland.