Abstract Objective—To evaluate the use of high-resolution MRI for hippocampal volumetry in dogs and to define a lower reference limit for hippocampal formation (HF) volume. Animals—20 dogs (with no history of seizures and no underlying structural brain disease) that underwent MRI of the brain. Procedures—The MRI protocol included a high-resolution T1-weighted 3-D ultrafast gradient-echo sequence aligned in a dorsal plane perpendicular to the long axis of the HF. Images obtained with MRI were retrospectively analyzed by 2 observers (A and B). Intraobserver and interobserver agreement were calculated with the Lin concordance correlation coefficient. Volume measurements of the HF were adjusted for intracranial volume, and a lower 95% reference limit for adjusted HF volume was calculated. Results—There was substantial intraobserver agreement (Lin concordance correlation coefficient, 0.97 [95% confidence interval {CI}, 0.94 to 0.99]) but poor interobserver agreement (Lin concordance correlation coefficient, 0.63 [95% CI, 0.37 to 0.79]). The lower 95% reference limit for adjusted HF volume was 0.56 cm³ (90% CI, 0.52 to 0.60 cm³) for the right HF and 0.55 cm³ (90% CI, 0.52 to 0.58 cm³) for the left HF. Conclusions and Clinical Relevance—HF volumes should be adjusted for intracranial volume to account for the large variation in canine skull size. The amount of time required to perform HF volumetry and low interobserver agreement may restrict this technique to research applications, such as the investigation of epileptic patients for hippocampal sclerosis or other cognitive disorders.