Background: HSP90B1, a member of the heat-shock protein 90 family, plays a vital role as a molecular chaperone for oncogenes and stimulates tumour growth. However, its role in various cancers remains unexplored. Methods: Using the cancer genome atlas, gene expression omnibus the Human Protein Atlas databases and various other bioinformatic tools, this study investigated the involvement of HSP90B1 in 33 different tumour types. Results: The over-expression of HSP90B1 generally predicted poor overall survival and disease-free survival for patients with tumours, such as adrenocortical carcinoma, bladder urothelial carcinoma, kidney renal papillary cell carcinoma, and lung adenocarcinoma. In this study, HSP90B1 was highly expressed in the majority of tumours. A comparison was made between the phosphorylation of HSP90B1 in normal and primary tumour tissues, and putative functional mechanisms in HSP90B1-mediated oncogenesis were investigated. Additionally, the mutation burden of HSP90B1 in cancer was evaluated along with the survival rate of patients with cancer patients. Conclusion: This first pan-cancer investigation reveals the oncogenic functions of HSP90B1 in various cancers.