Abstract The hippocampus is a critical structure for learning and memory formation injured by diverse neuropathologies such as epilepsy or Alzheimer's disease. Recently, clinical investigations have attempted to use diffusion tensor MRI as a more specific surrogate marker for hippocampal damage. To first better understand the tissue architecture of healthy hippocampal regions, this study characterized 10 rat hippocampi with diffusion tensor imaging (DTI) at 50-μm in-plane image resolution using a 14.1-T magnet. Chemical fixation of the dissected and straightened rat hippocampus provided a simple, effective way to reduce partial volume effects when segmenting hippocampal regions and improved mean signal-to-noise per unit time (e.g. 50.6 ± 4.4 at b = 1250 s/mm 2 in 27 min). Contrary to previous reports that water diffusion is homogeneous throughout the nervous system, statistically different mean diffusivities were observed (e.g. 0.238 ± 0.054 and 0.318 ± 0.084 μm 2/ms for the molecular and granule cell layers respectively) (ANOVA, P < 0.05). Different hippocampal subregions had lower fractional anisotropy than uniformly fibrous structures like corpus callosum because of their complex architecture. DTI-derived color fiber orientation maps and tractography demonstrated most components of the trisynaptic intrahippocampal pathway (e.g. orientations in stratum lacunosum–moleculare were dominated by perforant and Schaffer fibers) and also permitted some assessment of connectivity in the rat hippocampus.