Purpose: To report the detection of structural and functional biological changes in living animals using small animal in vivo MRI that complements traditional ex vivo histological techniques. We report the development and validation of the application of large deformation high dimensional mapping (HDM-LD) segmentation for the hippocampus in the rat. Materials and Methods: High resolution volumetric T2 weighted MRI images were acquired at 4.7 Tesla from six male in-breed nonepileptic Wistar rats. Two HDM-LD seg- mentations of the hippocampus (automated 1 and auto- mated 2) were compared with the manual segmentations of two investigators who independently segmented the hip- pocampi (manual 1 and manual 2). Results: The mean overlap for the hippocampi between automated 1 and automated 2 for the right hippocampi was 94.4% (SD 1.0) and for the left hippocampi was 94.3% (SD 2.5), while the mean overlap between automated 1 and manual 1 for the right hippocampi was 91.4% (SD 1.3) and for the left hippocampi was 91.9% (SD 1.4). Mean values for absolute differences for comparisons of all the segmenta- tions were the following: automated 1 versus automated 2, 3.2% (SD 1.0); manual 1 versus manual 2 6.82% (SD 5.22); automated 1 versus manual 1 13.0% (SD 1.8). Conclusion: HDM-LD can be applied to obtain accurate and reproducible three-dimensional segmentations of the hippocampus from rat MR images. HDM-LD will be a useful tool for investigations of hippocampal structural changes in vivo in rat models of human disease.