Magnetic resonance diffusion-based tractography techniques have offered a real breakthrough in brain studies. These methods allow, for the first time, to explore the anatomical organization of white matter pathways in humans, in-vivo and non-invasively. As any other method, diffusion-based tractography has limitations. The inherent limits related to the indirect measurement of the diffusion signal, and the strong dependence of this technique on acquisition, models and algorithm parameters, prevents the reliable reconstruction of some major white matter bundles. This dissertation targets the methods, limitations, improvements, and validation of tracking methods, with applications in neurobiological and clinical research. In particular, the main work focuses on a white matter bundle that represents a notable omission in tractography studies: the acoustic radiation (AR), a major projection sensory pathway conveying auditory information from the thalamus to the auditory cortex. Topographical knowledge of this bundle is scarce, and its in-vivo tractography reconstruction remains challenging, preventing the investigation of auditory and language functional mechanisms in humans. This dissertation investigates, for the first time, the topography of the AR using post-mortem blunt dissections and provides a detailed description of the trajectory of these fibres and their relationship with major neighbouring white matter bundles. The topographical information is then applied to conduct an investigation on the effects of different MRI acquisition and tractography parameters on the in-vivo tractography reconstruction of the AR. An optimal set of parameters is obtained for AR reconstructions and used to build the first tractography atlas of the acoustic radiation. The AR atlas is then applied to study congenital deaf patients. The optimized reconstruction parameters and the atlas generated in this dissertation may be used in future studies interested in identifying and characterizing the AR. The reliable 3D reconstruction of this bundle will improve our understanding of the functional mechanisms underlying hearing and language in healthy subjects and patients, as well as in neurosurgical applications.