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Diffuse axonal injury (DAI) is the predominant effect of severe traumatic brain injury and contributes significantly to neurological deficits. However, it is difficult to diagnose or characterize non-invasively with conventional imaging. Our study provides significant validation of a visual and statistical diffusion tensor imaging (DTI) technique as compared with pathological and electron microscopic study in a rat DAI model at multiple predilection sites and time points following trauma. Two DTI parameters, fractional anisotropy (FA) and axial diffusivity (AD), were significantly reduced from 12 h to 5 days post-trauma, corresponding to pathological axonal injury. At 7 days post-trauma, FA remained decreased, whereas AD pseudo-normalized and radial diffusivity increased. The temporal alterations in DTI parameters were observed in multiple predilection sites, and the extent of the changes in these parameters correlated significantly with the severity of histologically visualized axonal injury, as assessed by integrated optical density of immunochemically stained injured axons with quantitative stereology. Although anatomical T2-weighted magnetic resonance images showed no abnormal signals in microscopic lesions, we detected and characterized axonal injury directly by DTI at each time point. These results demonstrate that DTI has significant potential as a non-invasive tool with which to quantitatively diagnose and evaluate microstructural injury in the experimental and clinical assessment of DAI. This method can assist in accurate evaluation of the extent of axonal injury, detection of severe predilection foci, determination of approximate time of injury, and monitoring of the pathogenic condition at the early post-injury stage.