Background and Aims: Heat shock protein 90 (Hsp90) proteins constitute an important gene family of molecular chaperons. High-temperature stress, which is often combined with drought stress, may exert major constraints to grapevine growth and development. The aim of this study was to characterise the Vitisvinifera Hsp90 gene family. Methods and Results: Using the complete grapevine genome sequences, four cytoplasmic and three organelle-specific V. vinifera Hsp90 (VvHsp90) proteins were identified. Phylogenetic analysis revealed that they share high sequence similarity with their Arabidopsis counterparts, while the cytosolic isoforms are clustered into two distinct groups (VvHsp90.1 and VvHsp90.2). Transcriptional analysis showed that a representative gene from the first group (VvHsp90.1a), in contrast to VvHsp90.2a, is induced by heat shock in all vegetative tissues/organs tested. Interestingly, it was also expressed in tendrils in the absence of stress. The severity and duration of heat stress influenced in a complex manner the expression profile of VvHsp90.1a, while the other VvHsp90s tested were rather constitutively regulated. However, the endoplasmic reticulum-specific VvHsp90.7 was mildly and transiently induced by a relatively prolonged heat stress. Combined drought with heat stress resulted in a delay in VvHsp90.1a induction. Conclusions: Gene structure organisation and expression characteristics of VvHsp90s resemble those of their Arabidopsis orthologs, although species-specific differences also exist. Differential regulation of genes suggests functional diversification among isoforms. Significance of the Study: This is the first report on the characterisation of Hsp90s in grapevine. The present study contributes to a deeper understanding of the complex molecular responses of grapevine to stress.