BACKGROUND: The corticospinal tract (CST) is a major tract for motor function. It can be impaired by stroke. Its degeneration is associated with stroke outcome. Diffusion tensor imaging (DTI) tractography plays an important role in assessing fiber bundle integrity. However, it is limited in detecting crossing fibers in the brain. The crossing fiber angular resolution of intra-voxel structure (CFARI) algorithm shows potential to resolve complex fibers in the brain. The objective of the present study was to improve delineation of CST pathways in monkey brains scanned by conventional DTI. METHODS: Healthy rhesus monkeys were scanned by diffusion MRI with 128 diffusion encoding directions to evaluate the CFARI algorithm. Four monkeys with ischemic occlusion were also scanned with DTI (b = 1000 s/mm, 30 diffusion directions) at 6, 48, and 96 hours post stroke. CST fibers were reconstructed with DTI and CFARI-based tractography and evaluated. A two-way repeated MANOVA was used to determine significances of changes in DTI indices, tract number, and volumes of the CST between hemispheres or post-stroke time points. RESULTS: CFARI algorithm revealed substantially more fibers originated from the ventral premotor cortex in healthy and stroke monkey brains than DTI tractography. In addition, CFARI showed better sensitivity in detecting CST abnormality than DTI tractography following stroke. CONCLUSION: CFARI significantly improved delineation of the CST in the brain scanned by DTI with 30 gradient directions. It showed better sensitivity in detecting abnormity of the CST following stroke. Preliminary results suggest that CFARI could facilitate prediction of function outcomes after stroke.