White matter (WM) fiber bundles of the brain can be delineated by diffusion tractography utilizing anatomical regions-of-interest (ROI). These ROIs can specify seed regions in which tract generation algorithms are initiated. Interactive identification of such anatomical ROIs enables the detection of the major WM fiber tracts, but suffers from inter-rater and intra-rater variability, and is time consuming. We developed and compared three techniques for automated delineation of ROIs for the detection of two major WM fiber tracts in 12 healthy subjects. Tracts identified automatically were compared quantitatively to reference standard tracts derived from carefully hand-drawn ROIs. Based on comparative performance of the experimental techniques, a multi-template label fusion algorithm was found to generate tracts most consistent with the reference standard.