OBJECTIVE To investigate the utility of whole-brain diffusion tensor imaging (DTI) in elucidating the pathogenesis of multiple sclerosis (MS) using the normal-appearing white matter (NAWM) of the corpus callosum (CC) as a marker of occult disease activity. METHODS A high signal-to-noise ratio (SNR) and optimized entire brain DTI data were acquired in 26 clinically-definite relapsing and remitting multiple sclerosis (RRMS) patients and 32 age-matched healthy adult controls. The fractional anisotropy (FA) values of seven functionally distinct regions in the normal-appearing CC were compared between patients and controls. RESULTS This study indicates that 1) there was a gender-independent FA heterogeneity of the functionally specialized CC segments in normal volunteers; 2) FA in the MS group was significantly decreased in the anterior (P=0.0039) and posterior (P=0.0018) midbody subdivisions of the CC, possibly due to a reduction of small-caliber axons; and 3) the FA of the genu of the CC was relatively intact in the MS patients compared to the healthy age-matched controls (P=0.644), while the splenium showed an insignificant trend of reduced FA values (P=0.248). The decrease in FA in any of the CC subdivisions did not correlate with disease duration (DD) or the expanded disability status scale (EDSS) score. CONCLUSIONS The preliminary results are consistent with published histopathology and clinical studies on MS, but not with some published DTI reports. This study provides insights into the pathogenesis of MS, and the role played by compromised axonal integrity in this disease.