Medial glenohumeral ligament injury is commonly reported during medial shoulder joint instability in dogs. Arthroscopy is considered the gold standard procedure, but it is invasive and requires distension of the joint. Ultrasonographic examination of the medial glenohumeral ligament has been studied as a possible, less invasive alternative to arthroscopy however it has not been considered a useful method of assessment due to the interference of the probe with the pectoral muscles. The aims of this prospective analytical randomized pilot study were to develop a standardized ultrasound protocol for visualizing the canine medial glenohumeral ligament and to compare goniometry and ultrasound findings in cadaver dogs with versus without transection of the medial glenohumeral ligament. Nine adult Beagle cadavers (18 shoulders) were used. The first six shoulders were used in a preliminary study to describe an ultrasound technique to identify the medial glenohumeral ligament. Arthroscopy was performed on the remaining 12 shoulders, with six randomly selected medial glenohumeral ligaments from these shoulders, transected during the procedure. Ultrasound examination was performed after each arthroscopic procedure by an ultrasonographer blinded to the patient group. Four medial glenohumeral ligaments (67%) were correctly identified during the preliminary study. Ultrasonographic examination failed to diagnose the transection of all six medial glenohumeral ligaments in the second part of the study. No difference was observed in the ligament thickness between the dogs with and without a transected medial glenohumeral ligament. Dogs with a transected medial glenohumeral ligament had a wider articular space compared to dogs without a transected ligament (P < 0.001), and an articular space wider than 8.2 mm was discriminatory of a transected medial glenohumeral ligament in all the shoulders. In conclusion, the medial glenohumeral ligament could be identified with a medial ultrasonographic approach of the shoulder and a wider articular space can be a sign of a medial shoulder joint instability. Further studies are needed to confirm these preliminary findings in living dogs, with and without shoulder instability., (© 2022 American College of Veterinary Radiology.)