The aim of the present study was to establish which adrenal gland measurement was characterized by the least variations. To do this, we quantified the variability of seven different size measurements of the canine adrenal gland (maximal length, maximal height at the cranial and caudal poles on longitudinal and transverse images, and maximal width of the cranial and caudal poles) within observer, between observer, and between dogs based on three different measurements made by each of the three observers in six healthy Beagle dogs. The height of the caudal pole of both adrenal glands measured on longitudinal images had the lowest intra- and interobserver variability, while measurements of the length had the highest intra- and interobserver variability. Other measurements that were characterized by low intra- and interobserver variability were: height and width of the caudal pole on transverse images and height of the cranial pole on longitudinal images only. These results provide a basis for further study of the changes in adrenal gland size in dogs with pituitary-dependent hyperadrenocorticism.