Individuals with congenital monocular blindness are born without binocular vision and stereopsis, the effects of which on the brain microstructure are largely unknown. This study aims to investigate the microstructural characteristics of white matter tracts over the whole brain in congenital monocular blindness. We used T1 weighted MRI and diffusion tensor imaging (DTI) to investigate the microstructural characteristics of the brain in 16 patients with unilateral congenital microphthalmia (CM) and 16 matched normally sighted controls. The DTI-derived metrics were assessed using atlas-level analysis with FDR correction and TBSS-level analysis with threshold-free cluster enhancement correction (TFCE). CM exhibited significantly abnormal DTI-derived indices (p < 0.05, q < 0.05 of FDR correction) as follows: 1) declined fractional anisotropy (FA) in the inferior fronto-occipital fasciculus contralateral to the affected eye, bilateral inferior longitudinal fasciculus, while enhanced in the ipsilateral cingulum; 2) increased local diffusion homogeneity in the contralateral corticospinal tract while decreased in the ipsilateral superior longitudinal fasciculus; 3) reduced axial diffusivity (AD) in the body of corpus callosum. Meanwhile, the alteration tendencies of FA, AD, and radial diffusivity (RD) in the forceps major (increased FA and AD, decreased RD) and forceps minor (decreased FA and AD, increased RD) were interestingly opposite. These results reveal extensive microstructural abnormalities of WM ranging from sensory modalities to other cross-modal pathways involving language, execution, memory, emotion, fine movement, and interhemispheric communication as well. This study provides novel evidence of large-scale subcortical involvement subsequent to prolonged loss of half visual inputs, which may be associated with developmental delay and compensatory plasticity.