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OBJECTIVE: A new method of segmentation and fiber tractography of corpus callosum in diffusion tensor imaging (DTI) is provided, and applied in the study of children with high functioning autism. METHOD: Diffusion tensor imaging data has been used to analyze the fiber tractography of corpus callosum, which has been manually extracted out and subdivided into five subregions in the scheme proposed by Hofer. DTI data has been acquired from two male subjects aged 8, including one child diagnosed as high-functioning autism and one typically developing child as control. RESULTS: Depending on DTI-based fiber tractography, we distinguished five fiber bundles of corpus callosum projected into prefrontal, premotor, supplementary motor, primary motor, and primary sensory areas as well as into parietal, temporal, and occipital areas after segmentation of corpus callosum successfully. Topography of callosal fiber tracts has been used to analyze the cortical connectivity in DTI data and realized the fiber visualization which presents an efficient description of the white matter difference between the child with high functioning autism and the healthy control. CONCLUSIONS: DTI-based region-of-interest (ROI) fiber tractography of the entire corpus callosum and subregions provide a comprehensive and comparable method to study the white matter organization, particularly applied in autism.