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BACKGROUND AND PURPOSE:Tract-based spatial statistics (TBSS) has been used to assess the integrity of the visual pathway in glaucoma patients. TBSS uses the subjects' FA data to create a mean FA skeleton of white matter tracts before running voxel-wise cross-subject statistics. We compared four different approaches of registration of FA maps to create the skeleton and evaluated alignment and subsequently the impact of the chosen registration on voxel-wise statistics. MATERIAL AND METHODS:Our study comprised 69 subjects, i.e. 46 patients with primary open angle glaucoma (POAG) and a healthy, age-matched control group of 23 subjects. Mean FA skeletons were created using the following registration approaches: registration to a standard template (T), registration to the group mean (GM), registration to a group-wise atlas (GW) and registration to the most typical subject (N). Subsequently, maps of standard deviation of the 4D images were created to assess the alignment. Voxel-wise statistics for each registration approach were performed. RESULTS:We found distinct differences in voxel-wise statistics depending on the chosen registration approach. Best alignment results were achieved by registration to a study specific template, i.e. to the group mean (GM) or to a group-wise atlas (GW). Overall alignment did not differ between these two approaches. However, voxel-wise statistics showed clusters of significantly decreased FA values in the T and GM approach, which were not significant after GW registration. These voxels of significantly decreased FA values after T and GM registration did not represent white matter tracts and correlated with higher standard deviation in FA maps across subjects, thus implying registration errors, especially in the optic radiation. CONCLUSION:Registration to a study-specific template, i.e. to the group mean or a group-wise atlas seems to be the method of choice in TBSS-analysis of glaucoma patients as it shows better alignment of the optic radiation and helps to rule out registration errors due to misalignment.