This study proposes the use of the voxel-based morphometry (VBM) technique to investigate structural alterations of the cerebral cortex in patients with strabismus and amblyopia (SA). Sixteen patients with SA and sixteen healthy controls (HCs) underwent magnetic resonance imaging. Original whole brain images were analyzed using the VBM method. Pearson correlation analysis was performed to evaluate the relationship between mean gray matter volume (GMV) and clinical manifestations. Receiver operating characteristic (ROC) curve analysis was applied to classify the mean GMV values of the SA group and HCs. Compared with the HCs, GMV values in the SA group showed a significant difference in the right superior temporal gyrus, posterior and anterior lobes of the cerebellum, bilateral parahippocampal gyrus, and left anterior cingulate cortex. The mean GMV value in the right superior temporal gyrus, posterior and anterior lobes of the cerebellum, and bilateral parahippocampal gyrus were negatively correlated with the angle of strabismus. The ROC curve analysis of each cerebral region confirmed the accuracy of the area under the curve. Patients with SA have reduced GMV values in some brain regions. These findings might help to reveal the potential pathogenesis of SA and its relationship with the atrophy of specific regions of the brain.