The aim of this study was to investigate the molecular and protein expression pattern of markers of stemness phenotype and its clinicopathological significance in human biliary tract cancer (BTC). Human BTC cell lines (CCLP-1, Egi-1, MzChA-1, MzChA-2, SkChA-1, TFK-1 and GBC) were analyzed in vitro and in xenotransplanted animals for expression of markers of stemness and compared to tissue microarrays (TMA) of 34 cases of human BTC with complete pathomorphological and clinical data (survival). Molecular analyses on the mRNA and protein level included makers of stemness and progenitor (Bmi-1, Sox-2, Nestin, CD133, CD44 and Nanog), proliferation and differentiation (cell cycle proteins, intermediate filaments). The investigated BTC samples showed a low to moderate and partially significantly different expression pattern of the stem cell markers in vitro, in vivo and in TMA. Hierarchical cluster analysis identified subgroups with homogenous expression of stem cell markers significantly differing with respect to cytokeratin expression in xenografts and Ki67 proliferation marker in human TMA, respectively - thus indicating possible heterogeneous carcinogenesis pathways in BTC. Additionally, these stem cell markers could be linked to morphology and molecular markers of proliferation and differentiation on the mRNA and protein level. Finally, survival analysis identified the combination of CD133 and CD44 as an independent prognostic factor yet their value as prognostic factors need testing in prospective study design.