High temperature during grain filling stage in wheat is greatly affects the wheat production by altering its physiological and biochemical process. Understanding the response of physio-biochemical traits of wheat genotypes under late sowing heat stress condition will facilitate the development of high yielding, stress-tolerant wheat genotypes. Eight diverse wheat cultivars were used to characterize the physiological and biochemical traits for the improvement of heat tolerance by using heat tolerance index (HTI), heat susceptibility index (HIS), multivariate analysis and heatmapping. The flag leaf proline content and water-soluble carbohydrate (WSC) accumulation were increased by 19.0% and 38.9% respectively, while in kernel, the proline was decreased by 23.5% from the optimum to late grown heat condition. The principal component analysis and biplot analysis showed that initial principal components, PC1 and PC2 represented 83% of total cumulative variation. The chlorophyll content, yield and yield contributing characters, proline and WSC showed significant differences between the heat tolerant and heat susceptible variety. Among the studied cultivars, BARI GOM-30 performed better than the others in heat stressed condition. The cluster analysis demonstrated the cultivars were categorized into three groups and Spk and Gr seem to be interesting traits as well as HTI and HIS. It’s concluded that the physio-biochemical traits associated with HTI for grain yield are the major factors which may contribute in improved wheat productivity. If these traits performed appropriately under stress condition the crop will show more productive returns under changing climate.