show Abstracthide AbstractGallbladder cancer (GBC) has the worst survival among diverse biliary tract cancers (BTCs). However, no molecular-based therapies are yet approved for GBC. The current molecular characterization and known molecular markers are insufficient, necessitating further evaluation of the genome and mRNA expression data of GBCs. To characterize the molecular features of GBCs and compare them with other BTC types, here we performed a comprehensive analysis of the expression of RNAs, including ncRNAs, in GBC and hilar bile duct cancers. Although the transcriptomic difference based on tumor location in the bile duct was unclear, we could classify GBCs into two subclasses based on the tumor microenvironment and patient prognosis. We also identified the alterations in the expression of some ncRNAs and genes related to tumor microenvironment, such as those related to EMT, tumor immunology, and transforming growth factor (TGF)-beta signaling pathway. This comprehensive analysis may elucidate GB carcinogenesis and provide a new classification of GBCs that may be useful for therapeutic decision-making. Overall design: Expression profiling of RNA and ncRNA of 36 GBCs and eight hilar bile duct cancers.