Magnetic resonance imaging (MRI) has transformed the world of non-invasive imaging for diagnostic purposes. Modern techniques such as diffusion weighted imaging (DWI), diffusion tensor imaging (DTI), and diffusion spectrum imaging (DSI) have been used to reconstruct fiber pathways of the brain - providing a graphical picture of the so-called "connectome." However, there exists controversy in the literature as to the accuracy of the diffusion tractography reconstruction. Although various attempts at histological validation been attempted, there is still no 3D histological pathway validation of the fiber bundle trajectories seen in diffusion MRI. Such a validation is necessary in order to show the viability of current DSI tractography techniques in the ultimate goal for clinical diagnostic application. This project developed methods to provide this 3D histological validation using the rhesus monkey motor pathway as a model system. By injecting biotinylated dextran amine (BDA) tract tracer into the hand area of primary motor cortex, brain section images were reconstructed to create 3D fiber pathways labeled at the axonal level. Using serial coronal brain sections, the BDA label was digitized with a high resolution digital camera to create image montages of the fiber pathway with individual sections spaced at 1200 micron intervals through the brain. An MRI analysis system, OSIRX, was then used to reconstruct these sections into a 3D volume. This is an important technical step toward merging the BDA fiber tract histology with diffusion MRI tractography of the same brain, enabling identification of the valid and inaccurate aspects of diffusion fiber reconstruction. This will ultimately facilitate the use of diffusion MRI to quantify tractography, non-invasively and in vivo, in the human brain.