Purpose: Investigation of stroke lesions mostly focuses on the grey matter (GM). White matter (WM) degeneration during acute stroke has remained understudied. In the present study, monkeys were employed to investigate the alterations in GM and WM in the brain following ischemic occlusion using diffusion tensor imaging (DTI). Materials and Methods: Permanent middle cerebral artery occlusion was induced in rhesus monkeys (n = 6) using an interventional approach. Serial DTI was conducted on a clinical 3 T in the hyperacute phase (2–6 hours), 48, and 96 hours post-occlusion. Regions of interest in GM and WM of lesion areas were selected for data analysis. Results: Mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD) in WM decreased substantially during hyperacute stroke, similar to those seen in GM. No obvious fractional anisotropy changes were seen in WM during the hyperacute phase until 48 hours poststroke when significant fiber loss was observed. Pseudo-normalization of MD, AD, and RD was seen at 96 hours. Pathological changes in WM and GM were observed in ischemic areas at 8, 48, and 96 hours poststroke. Relative changes in MD, AD, and RD of WM were correlated negatively with infarction volumes at 6 hours poststroke. Conclusion The present study revealed the microstructural changes in GM and WM of monkey brains during acute stroke using DTI. The preliminary results suggest that AD and RD may be sensitive surrogate markers to assess specific microstructural changes in WM during the hyperacute stroke.