The gallbladder is routinely evaluated during ultrasonographic examinations in dogs. However, published studies describing the effects of sedative agents on gallbladder wall thickness are currently lacking. The aims of this prospective, blinded, randomized crossover pilot study were to test hypotheses that IV morphine would result in gallbladder wall thickening, that morphine administration would increase plasma histamine concentrations, and that combining IV morphine with dexmedetomidine would potentiate gallbladder wall thickening. Six healthy Beagle dogs were sedated with intravenous (IV) morphine 0.4 mg/kg (group M), dexmedetomidine 7 μg/kg (group D), or a combination of the two (group MD). Physiologic parameters were measured at baseline and at regular intervals until the last ultrasonographic scan. Ultrasonographic scans were performed at baseline, 90 s, and at 5, 15, 30, 45, 60, 90, and 120 min. Plasma histamine samples were taken at baseline, 90 s, and 5 and 60 min. Cochran's Q-test was used to compare gallbladder wall thickening between groups, while the association between histamine plasma concentration and gallbladder wall thickness was compared with a mixed-effects model. Baseline gallbladder wall thickness was not significantly different between groups. Six of 18 treatments/dogs (33%) developed gallbladder thickening, with no difference between groups. There was no significant difference in baseline plasma histamine concentrations between groups, and no association between plasma histamine concentration and gallbladder wall thickness. Gallbladder wall thickening was observed in at least one dog in each group, therefore caution is recommended for gallbladder wall thickness ultrasonographic interpretation in dogs when these drugs have been administered.