Wheat is a globally important crop used as a main staple food in various countries of the world. The current study was conducted with the objective to evaluate the effect of a high temperature (HT) on osmolytes (starch, sucrose, total soluble sugars, total soluble proteins and proline), physiological parameters (Chl-a, Chl-b, photosynthesis rate, transpiration rate and stomatal conductance), antioxidant enzymes (superoxide dismutase, catalase and peroxidase) and agronomic traits (flag leaf area, spike length, and thousand grain weight) during the grain filling and anthesis stages of wheat cultivars (Fakhr-e-Bhakar, Raj-3765, Jimai-22 and Bayraktar-2000) collected from different regions of the world. Separate experiments for both stages were conducted in a glasshouse and treated with two different temperature regimes, i.e., optimum (OT) (24 °C day; 14 °C night) and high temperature (HT) (32 °C day; 22 °C night) in RCBD for two weeks. The data for osmolytes, antioxidant enzymes and physiological contents were collected at days 3, 5, 7, 9 and 13 after the start of plant stress, while the agronomic traits were collected at maturity. The data obtained were subjected to a statistical analysis using the statistix8.1 and R-program. HT stress significantly reduced all the traits except for the membrane damage, transpiration rate, proline and total soluble sugars, whose values increased considerably in the genotype Bayraktar-2000. However, under both regimes of temperature Fakhr-e-Bhakkar showed a high tolerance against HT stress, as revealed by physiological, biochemical and agronomic evaluations. Moreover, correlation, PCA and heat map analyses indicated that all types of traits are significantly interconnected in determining the crop potential to sustain its growth under HT stress.