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Statistical analysis on diffusion tensor imaging has been used extensively in mesial temporal lobe epilepsy (mTLE) and most studies report decrease in fractional anisotropy (FA) in multiple white matter regions. However, these findings vary across studies and between regions. Therefore, in this study, we used tract-based spatial statistics along with machine learning approaches to investigate the whole-brain white matter changes between 17 left mTLE patients and 15 right mTLE patients and 34 matched healthy controls. The results showed that the three groups could be distinguished from each other with promising accuracy. Compared with controls, the FA value of the most discriminating voxels was decreased in the ipsilateral limbic system, corpus callosum, and temporal white matter in both patient groups. Compared with right mTLE, left mTLE had decreased FA in the left temporal white matter, whereas right mTLE had decreased FA in the right frontal and temporal white matter, and right posterior corona radiata. These findings not only provide useful information for lateralization of the seizure focus but can also be used as a potential biomarker for the diagnosis and treatment of the mTLE. This may be helpful in assessment of patients with mTLE when no lesion is detected on visual evaluation.