To demonstrate the feasibility of in-vivo delineation of corticospinal tracts of non-human primates, high angular resolution diffusion imaging (HARDI) tractography¹ was utilized to examine the corticospinal tracts distribution for sub-regions of motor cortices of adult macaque monkeys. Five healthy rhesus monkeys (female, ~10 years old) were utilized. The HARDI pulse sequence was custom-developed and implemented under the software IDEA on a Siemens 3.0T TIM Trio scanner. Diffusion images were acquired with a Siemens 8-channel phased-array volume coil and a dual spin-echo, echo planar imaging (EPI) sequence and the following imaging parameters: TE = 107 ms, TR = 6000 ms, data matrix = 83 × 83, voxel size = 1.3 mm × 1.3 mm × 1.3 mm. HARDI data were collected at a single b-value of 2000 s/mm² with 128 diffusion encoding directions chosen to be approximately isotropically distributed on a sphere according to the electrostatic repulsion model. The motor cortical areas, including supplementary motor area (SMA), dorsal and ventro premotor cortex (dPMC and vPMC), primary motor cortex (PMC), primary somatosensory cortex (PSSC), posterior parietal cortex (PPC) and spinal cord defined from a monkey brain template were registered to the B0 images using the FSL software (FMRIB, Oxford). The white matter tracts were reconstructed using DSI-Studio software (http://dsi-studio.labsolver.org) ². Corticospinal tracts for each motor area in both hemispheres was determined by using the motor area as a seed and terminating in the spinal cord, with 2000 seeds for each motor area for tracts calculation. The distribution of corticospinal tracts was then evaluated as the proportion of the number of corticospinal tracts for each motor area over the number of the entire motor areas. As seen in Figure 1, corticospinal tracts that originate from sub-regions of motor areas and terminate in the spinal cord were successfully delineated by HARDI tractography. Figure 2 showed the proportional corticospinal fibers from the sub-regions of motor areas. Most of the corticospinal fibers for the motor areas were associated with PMC and secondly from PSSC, while the least are from vPMC. The corticospinal fibers from SMA, dPMC, vPMC, PSSC and PPC were significantly different from that originating from PMC (p < 0.05). With HARDI tractography, it was shown that the most proportion of corticospinal fibers was from PMC (i.e., 36% of the total corticospinal fibers), and less proportions from the other motor areas anterior or posterior to PMC (i.e, 32% from SMA, dPMC and vPMC, and 31% from PSSC and PPC). These results are similar to the previous ex-vivo macaque results where 30% of corticospinal fibers arise from PMC, 30% of corticospinal fibers from SMA and the premotor cortices and 40% of corticospinal fibers supply the rest ³. Given the similarity of the present in-vivo study to the prior ex-vivo findings in macaque brains, our results suggest that HARDI tractography provides an unprecedented way for in-vivo corticospinal fibers characterizations in potential macaque monkey models mirroring corticospinal tract degenerations ⁴. Our results also showed high similarity in PMC (37%) and dPMC (10%) but more difference in SMA (21%) and PSSC (32%) of human brain by in-vivo DTI tractography, suggesting regional distinctiveness between the two species ⁵.