Heat stress causes an array of physiological, biochemical and morphological changes in plants, affecting growth and yield. Here, we report cloning of HSP90 gene of 2,323 bp from C-306 cultivar of wheat having ORF from 62 to 2,164 bp encoded for 700 amino acids. Quantitative real time expression analysis of HSP90 gene in C-306 showed 1.5, 1.2, 2.5 fold (in root), 4.5, 4.3 and 6.5 fold increase (in flag leaf) in the transcript level at pollination, milky dough and seed hardening stages. HSP90 transcript level was observed low in root as well as shoot of susceptible cultivar (PBW343) at different stages of growth. A significant difference in the fold expression of HSP90 was observed in C-306 and PBW343 against differential heat shock. An altered expression of H2O2 and decline in proline accumulation was observed in C-306 at different stages of growth. Western blot analysis revealed the presence of 5, 6 and 5 multiprotein chaperone complexes of HSP90 in the range of 95 Da to 70 KDa at pollination, milky dough and seed hardening stages. An expression of few novel isoenzymes of superoxide dismutase and catalase was observed against differential heat shock. A decrease in cell membrane stability was observed at different stages of growth in C-306 cultivar of wheat. In conclusion, we suggest that a high HSP90 transcript level along with high activities of antioxidant isoenzymes and low proline accumulation is a promising target for developing wheat genotypes with tolerance to heat stress.