HSP90 is a key component in maintaining intracellular homeostasis, and it controls multiple cellular processes by regulating the maturation, stability, activity and turnover of its substrates. Rubber tree (Hevea brasiliensis Müll. Arg.) is an important source for the production of natural rubber, which has thousands of industrial uses and a high socioeconomic value. Using PCR technique, an HSP90 family member gene, named HbHSP90.6 gene (GenBank accession number: OP375588), was cloned from rubber tree variety Reyan73397 latex, and the sequence analysis showed that its coding region (CDS) was 2112 bp. Physicochemical analysis showed that its relative molecular weight was 80 774.85 Da, isoelectric point was 5.04, instability coefficient was 38.98, and total average hydrophilicity was -0.610, presumably HbHSP90.6 protein is a stable hydrophilic protein. The results of conserved structural domain analysis and multiple sequence alignment showed that HbHSP90.6 contained a structural domain of PTZ00272 and a highly conserved MEEVD motif, which is a member of the HSP90 family. Phylogenetic analysis showed that HbHSP90.6 clustered with Arabidopsis AtHSP90.1 (NM_124642.4) and rice OsHsp90-1 (LOC_Os04g01740) with 92.34% and 86.44% homology, respectively. Tissue expression analysis showed that the gene expression of HbHSP90.6 gene in latex was significantly higher than that in root, flower, branche, stem and leaf, and the expression of HbHSP90.6 gene in latex reached 283-fold relative to flowers, presumably the HbHSP90.6 gene may be involved in intracellular transport of latex tubes and regulation of latex metabolism. Mechanical wounding treatment in latex significantly upregulated the expression of HbHSP90.6 gene by 19 fold compared to the pretreatment pair, and it is hypothesized that HbHSP90.6 gene is involved in mechanical wounding regulated physiological and metabolic activity responses in rubber trees. The expression of HbHSP90.6 gene reached the highest level at 6 h of treatment with the phytohormones ethephon, jasmonic acid and 3-Indoleacetic acid, which were 45 fold, 17 fold and 6 fold higher than before treatment, respectively. The expression of HbHSP90.6 gene was significantly upregulated at 12 h after brassinosteroids treatment, reaching 50 fold of the pretreatment expression, presumably the HbHSP90.6 gene is involved in phytohormone mediated transcriptional regulation. Subcellular localization was performed by constructing a 35S::HbHSP90.6::GFP fusion expression vector, and the results showed that the HbHSP90.6 gene was localized in the cytoplasm and nucleus, which was consistent with the predicted results of subcellular localization. The results of this study would provide a thorough basis for elucidating the molecular mechanisms of HbHSP90.6 gene in plant hormone signaling-regulated stress and latex metabolism in rubber trees.