Systematic gait analyses have been widely used in clinical settings as a reliable means of evaluating stroke severity and the efficacy of rehabilitation on people. However, the extent of gait changes post-stroke in experimental quadrupeds remains to be explored. To date, gait studies in cerebral ischemia have been limited to the mild ischemia-reperfusion model. However, studies on pathophysiology and therapy of experimental stroke suggest that permanent middle cerebral artery occlusion (pMCAO) is more similar to naturally occurring cerebral ischemia in humans. This is the first preclinical study to demonstrate that pMCAO rats can be used to assess long-term functional deficits related to gait by a computer-assisted method. Our gait analysis results demonstrate obvious gait deficits in the acute phase of the disease. During recovery, gait function gradually improved, but deficits were still detectable 42 days post-pMCAO. Objective and accurate photogrammetric parameters were used to illuminate laws of impairment and compensation in rats at different stages of cerebral ischemia in injured and uninjured limbs during walking. Compared to previous gait studies involving transient (t) MCAO rats, gait changes observed in pMCAO rats were more similar to changes following naturally occurring cerebral ischemia in humans. Importantly, the average body rotation and propulsion index, not previously used, are specific parameters for accurately assessing gait function during the acute phase of post-pMCAO. Furthermore, the gait test results revealed significant correlations between the final infarction volume and earlier behavioral outcomes. In conclusion, the gait analysis is a promising tool for assessing cerebral ischemia severity, and that it may provide a new means of investigating mechanisms of cerebral ischemia and evaluating potential therapies.