Diffusion tensor magnetic resonance imaging can acquire quantitative information on the microstructural integrity of white matter structures and depict brain connectivity in vivo based on the behavior of water diffusion. Diffusion tensor imaging-derived tractography has been used for virtual dissection of the fornix and cingulum in healthy subjects, but not in patients with temporal lobe epilepsy (TLE). Eight patients with medically intractable TLE and unilateral mesial temporal sclerosis and nine healthy control subjects were imaged using diffusion tensor imaging. Fiber tracking was performed to delineate the fornix and cingulum, which were quantitatively analyzed. Bilateral symmetrical reduction in fractional anisotropy was observed in the fornix of patients with TLE, together with an increase in water mobility perpendicular to the axis of the fibers. The findings in the cingulum are similar to those of the fornix with the exception of significantly increased bulk diffusivity in the latter. We observed strikingly symmetrical bilateral abnormalities of axonal integrity in the fornix and cingulum in a series of patients with unilateral mesial temporal sclerosis. Our findings suggest that TLE with unilateral mesial temporal sclerosis is associated with bilateral limbic system pathology.