This study aimed to observe the microstructural alterations in corticospinal tract (CST) after motor pathway infarction and predict early Wallerian degeneration based on automated fiber quantification (AFQ). 53 patients with first-onset stroke in motor pathway and 29 health age-matched controls were enrolled. FA, MD, AD and RD values were significantly reduced on lesions of the affected side, while DKI values (MK, AK and RK) exhibited significant increase. AFQ was performed to identify differences on the whole CST pathway in the affected side between control and patient group. AD and MD values in CST of the affect side were significantly higher than them in healthy control. The findings of AD and MD have the same pathological changes on the affected CST pathway no matter the primary stroke lesions located in any regions (brainstem, posterior limb of internal capsule or above centrum semiovale). Our findings suggest that AFQ has the potential to detect the early Wallerian degeneration in the central nervous system in vivo after the first 24 hours in stroke.