Objective <p>The corpus callosum (CC) is the largest commissural white matter structure in the human brain, responsible for interhemispheric communication. Despite its clinical significance in neurosurgical procedures, detailed anatomical studies of its fiber organization remain limited. The aim of this study was to investigate the fiber architecture and topography of the CC through combined microsurgical dissection, tractography, and illustrative analysis.</p> Methods <p>Ten formalin-fixed adult human brains (20 hemispheres) were prepared using Klingler’s method. Fiber dissections were performed under an operating microscope in medial-to-lateral, superior-to-inferior, and lateral-to-medial orientations. High-resolution diffusion data from the Human Connectome Project were used for tractographic validation via DSI Studio. The findings were further supported by schematic illustrations.</p> Results <p>Dissection revealed a three-layered organization of callosal fibers: dorsal, intermediate, and ventral layers. Dorsal fibers connected homotopic cortical areas, particularly the motor and premotor cortices. Intermediate fibers projected toward the corona radiata and internal capsule. Ventral fibers included tapetal connections and fibers linking subcortical structures such as the caudate nuclei. Additionally, we identified accessory callosal bundles and cingular-splenial fibers, which have seldom been emphasized in the previous literature. Tractographic results confirmed the trajectories and lamination patterns observed during dissection.</p> Conclusion <p>Our integrative analysis provides a comprehensive educational depiction of corpus callosum lamination and topography. These findings should enhance anatomical understanding and inform neurosurgical approaches, particularly in callosotomy and transcallosal tumor surgery. Further studies are needed to explore the functional and clinical implications of these anatomical subdivisions.</p>