Injuries to the biliary tree during surgical, endoscopic or invasive radiological diagnostic or therapeutic procedures involving the pancreas, liver or organs of the upper gastrointestinal tract give rise to the need to develop a method for clear discrimination of biliary anatomy from surrounding tissue. Hyperspectral imaging (HSI) is an emerging optical technique in disease diagnosis and image-guided surgery with inherent advantages of being a non-contact, non-invasive, and non-ionizing technique. HSI can produce quantitative diagnostic information about tissue pathology, morphology, and chemical composition. HSI was applied in human liver transplantation and compared to porcine model operations to assess the capability of discriminating biliary anatomy from surrounding biological tissue. Absorbance spectra measured from bile ducts, gall bladder, and liver show a dependence on tissue composition and bile concentration, with agreement between human and porcine datasets. The bile pigment biliverdin and structural proteins collagen and elastin were identified as contributors to the bile duct and gall bladder absorbance spectra.