Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types which arise from epithelial cells of the pancreatobiliary system. Due to their histologic and morphologic similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. Biomarkers with high specificity and sensitivity for the differentiation of these tumour types would therefore be a valuable tool, but so far none are available. Here, we address this problem by comparing microdissected CCC and PDAC tumour cells in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of primary and secondary tumour tissue.