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There are seven distinct β-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. βV-tubulin, like βIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human βV-tubulin. The antibody did not cross-react with mouse βV-tubulin or other human β-tubulin isotypes and specifically labeled βV-tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that βV-tubulin was expressed in endothelial cells, muscle and cells with muscle differentiation, structures with transport and/or secretary function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretary function such as prostate. βV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. In a small number of malignancies, breast, lung and ovarian cancers, βV-tubulin was aberrantly expressed, suggesting that this isoform may be associated with tumorigenesis. Thus, βV-tubulin expression is a potentially promising prognostic marker of malignancy.