Background: Melatonin is a hormone produced and secreted primarily by the pineal gland and mainly metabolised in the liver. Increased melatonin concentrations have been reported in human cirrhosis and hepatic encephalopathy (HE), a syndrome of neurologic dysfunction. The pathogenesis of canine HE is incompletely understood. Melatonin has been hypothesised as a contributor to the development of HE. The aim of this study was to investigate whether serum melatonin concentrations are increased in canine congenital portosystemic shunting (cPSS), with and without HE. Methods: Medical records were retrospectively reviewed, for which archived (-80ºC) serum samples were available. A canine competitive ELISA was used to measure melatonin in two cohorts: dogs with a final diagnosis of cPSS (n=23) and healthy dogs (n=15). Results: Melatonin concentrations were not significantly different (P=0.81) between healthy controls (median 27.2pg/mL, range 19.8-161.5pg/mL) and dogs with cPSS (median 25.7pg/mL, range 18.5-244.9pg/mL). Serum melatonin did not differ between cPSS patients with and without clinical signs of HE (P>0.99). No correlation was found between serum melatonin and blood ammonia (rs=-0.41, P=0.08). Conclusion: Serum melatonin is not increased in canine cPSS with and without HE. We found no evidence that altered melatonin metabolism plays a role in the pathogenesis of cPSS-associated HE.