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Previous studies have demonstrated that strabismus or amblyopia can result in marked brain function and anatomical alterations. However, differences in spontaneous brain activity in strabismus and amblyopia (SA) patients as compared with control individuals remain unclear. The present study aimed to analyze the potential brain activity changes in SA patients and their association with behavioral performance. In total, 16 patients with SA (10 women and 6 men) and 16 healthy controls (HCs; 6 men and 10 women) with matched age and sex were recruited. All subjects were examined with resting-state functional magnetic resonance imaging (rs-fMRI), and changes in the spontaneous brain activity of SA patients were evaluated by the regional homogeneity (ReHo) method. The diagnostic ability of the ReHo method was assessed using receiver operating characteristic (ROC) curve analysis. In addition, the association between the mean ReHo value in different brain regions and the behavioral performance was explored by correlation analysis. It was observed that the ReHo value was significantly increased in SA patients compared with HCs in the following brain regions: left lingual gyrus, right middle occipital gyrus/precuneus, bilateral anterior cingulate, left middle occipital gyrus and bilateral precentral gyrus. By contrast, the ReHo value of the left inferior frontal gyrus was significantly lower than that in HCs. ROC curve analysis indicated that the ReHo method has certain credibility for the diagnosis of SA patients. In addition, no similar changes were detected in other brain regions. These results revealed abnormal spontaneous brain activity in certain parts of the brain in adult patients with SA, which suggests the involvement of the neuropathological or compensatory mechanism in these patients, and may be beneficial for clinical treatment. [ABSTRACT FROM AUTHOR]