Diffusion Tensor Imaging (DTI) is being increasingly used to assess white matter integrity and it is therefore paramount to address the test–retest reliability of DTI measures. In this study we assessed inter- and intra-site reproducibility of two nominally identical 3 T scanners at different sites in nine healthy controls using a DTI protocol representative of typical current “best practice” including cardiac gating, a multichannel head coil, parallel imaging and optimized diffusion gradient parameters. We calculated coefficients of variation (CV) and intraclass correlation coefficients (ICC) of fractional anisotropy (FA) measures for the whole brain, for three regions of interest (ROI) and for three tracts derived from these ROI by probabilistic tracking. We assessed the impact of affine, nonlinear and template based methods for spatially aligning FA maps on the reproducibility. The intra-site CV for FA ranged from 0.8% to 3.0% with ICC from 0.90 to 0.99, while the inter-site CV ranged from 1.0% to 4.1% with ICC of 0.82 to 0.99. Nonlinear image coregistration improved reproducibility compared to affine coregistration. Normalization to template space reduced the between-subject variation, resulting in lower ICC values and indicating a possibly reduced sensitivity. CV from probabilistic tractography were about 50% higher than for the corresponding seed ROI.