The aim of this study is to propose methods for assessing the reproducibility of diffusion tractography algorithms in future clinical studies and to show their application to the tractography algorithm developed in our unit, fast marching tractography (FMT). FMT estimates anatomical connectivity between brain regions using the information provided by diffusion tensor imaging. Three major white-matter pathways were investigated in 11 normal subjects - anterior callosal fibers, optic radiations, and pyramidal tracts. FMT was used to generate maps of connectivity metric, and regions of voxels with highest connectivity metric to an anatomically defined starting point were identified for each tract under investigation. The reproducibilities of tract-"normalized" volume (NV) and fractional anisotropy (FA) measurements were assessed over such regions. The values of tract volumes are consistent with the postmortem data. Coefficients of variation (CVs) for FA and NV ranged from 1.7 to 7.1% and from 2.2 to 18.6%, respectively. CVs were lowest in the anterior callosal fibers (range: 1.7- 7.8%), followed by the optic radiations (range: 1.2-18.6%) and pyramidal tracts (range: 2.6-15.5%), suggesting that fiber organization plays a role in determining the level of FMT reproducibility. In conclusion, these findings underline the importance of assessing the reliability of diffusion tractography before investigating white matter pathology. © 2003 Elsevier Science (USA). All rights reserved.