Background: Thickened corpus callosum is a rare finding and its pathophysiology is not well known. An anomalous supracallosal bundle has been depicted by fiber tracking in some cases but no diffusion tensor imaging metrics of thickened corpus callosum have been reported. Objective: To use diffusion tensor imaging (DTI) in cases of thickened corpus callosum to help in understanding its clinical significance. Materials and methods: During a 7-year period five children (ages 6months to 15years) with thickened corpus callosum were studied. We determined DTI metrics of fractional anisotropy (FA), mean diffusivity, and axial (λ1) and radial (λ2, λ3) diffusivity and performed 3-D fiber tracking reconstruction of the thickened corpus callosum. We compared our results with data from the literature and 24 age-matched controls. Results: Brain abnormalities were seen in all cases. All children had at least three measurements of corpus callosum thickness above the 97th percentile according to age. In all children 3-D fiber tracking showed an anomalous supracallosal bundle and statistically significant decrease in FA (P = 0.003) and λ1 (P = 0.001) of the corpus callosum compared with controls, but no significant difference in mean diffusivity and radial diffusivity. Conclusion: Thickened corpus callosum was associated with abnormal bundles, suggesting underlying axonal guidance abnormality. DTI metrics suggested abnormal fiber compactness and density, which may be associated with alterations in cognition