The anatomy of the avian gastrointestinal (GI) tract is uniquely suited to each species' dietary requirements. African penguins (Spheniscus demersus) are charismatic and popular exhibit animals. As their prevalence grows, there is a need to understand their unique digestive tract to diagnose abnormalities. Reference material specific to the digestive tract of piscivores is scant, and knowledge of the GI tract of a healthy penguin is based on information from other birds. The purpose of this study is to determine the normal gross anatomy, transit time, and histopathologic structures of the penguin GI tract. Twelve clinically healthy penguins were selected for this study from the colony at the Maryland Zoo in Baltimore, which, at the time of this study, consisted of 55 birds. All penguins underwent a barium contrast study, and radiographic images were obtained until the entire GI tract was empty. Approximately 2 wk later, each penguin was anesthetized, and an endoscopic evaluation of the anterior GI tract was performed. Time from barium administration to defecation ranged from 17 to 70 min, and on average, barium clearance was 17.6 hr (range, 5–36 hr). Fluid from the ventriculus had an average pH of 2.75 and contained a mixed bacterial population. Koilin presence and thickness appreciated on endoscopy did not correspond with the thickness determined on histopathology. The results of this study provide a comparative baseline to use during diagnostic workups and help guide treatment decisions.