Imaging and delineation of the optic radiations (OpRs) remains challenging, despite repeated attempts to achieve reliable validated tractography of this complex structure. Previous studies have used varying methods to generate representations of the OpR which differ markedly from one another and, frequently, from the OpR's known structure. We systematically examined the influence of a key variable that has differed across previous studies, the tractography seed region, in 13 adult participants (nine male; mean age 31 years; SD 8.7 years; range 16–47). First, we compared six seed regions at the lateral geniculate nucleus (LGN) and sagittal stratum based on the literature and known OpR anatomy. Three of the LGN regions seeded streamlines consistent with the OpR's three “bundles,” whereas a fourth seeded streamlines consistent with each of the three bundles. The remaining two generated OpR streamlines unreliably and inconsistently. Two stratum regions seeded the radiations. This analysis identified a set of optimal regions of interest (ROI) for seeding OpR tractography and important inclusion and exclusion ROI. An optimized approach was then used to seed LGN regions to the stratum. The radiations, including streamlines consistent with Meyer's Loop, were streamlined in all cases. Streamlines extended 0.2 ± 2.4 mm anterior to the tip of the anterior horn of the lateral ventricle. These data suggest some existing approaches likely seed representations of the OpR that are visually plausible but do not capture all OpR components, and that using an optimized combination of regions seeded previously allows optimal mapping of this complex structure. Hum Brain Mapp 35:683–697, 2014. © 2012 Wiley Periodicals, Inc.