Cholelithiasis is a rarely reported condition in lions and zoological carnivora. This report documents two geriatric, male, African lions (Panthera leo
Lions A and B) submitted for routine necropsy following euthanasia due to unrelated conditions. Pre-euthanasia serum biochemistry revealed unremarkable hepatobiliary markers for both lions. On necropsy, cholelithiasis was observed in both individuals, with Lion A displaying more severe gallstone accumulation and a biliary cystadenoma. Gallstones from Lion A were analysed using X-ray diffraction, element content analysis, and scanning electron microscopy. Findings revealed that the black appearance of the choleliths, the absence of acicular cholesterol, and minimal calcium content (1.05%) were consistent with bilirubin polymer composition. Risk factors for cholelithiasis in these lions may have included geriatric age, sex (male), good body condition, and chronic biliary tract inflammation. This case report presents the first documented instances of incidental cholelithiasis in African lions, providing novel findings into its composition (bilirubin polymers), possible risk factors, and histopathological features.