Heat stress alters photosynthetic components and antioxidant scavenging system, negatively affecting plant growth and development. Plants overcome heat stress damage through an integrated network involving enzymatic and non-enzymatic antioxidants. The aim of the study was to assess physiological and biochemical responses in contrasting thermotolerant wheat varieties exposed to 25°C (control) and 35°C (heat stress), during seedling stage. Our results revealed a substantial decrease in the photosynthetic pigments, carotenoids, anthocyanin content, and increased membrane injury index, malondialdehyde, lipoxygenase, methylglyoxal and H2O2 contents compared to non-stress wheat seedlings. Comparatively the heat tolerant variety BG26 maintained a high level of stability compared to the heat susceptible variety Pavon, perpetuated by higher accumulation of proline, glycine betaine, ascorbate-glutathione cycle associated enzymes, reduced glutathione and ascorbate contents. In addition, significantly lower MG detoxification and activities of antioxidant system and ascorbate-glutathione cycle related enzymatic activities lead to increased susceptibility in Pavon. Hierarchical clustering and principal component analysis revealed BG26 possessing a combination of biochemical responses that induced higher level of tolerance. Taken together, our results provide a reference for utilizing BG26 and Pavon as highly contrasting heat-responsive varieties for comparative genomics and translational research to unravel underlying mechanisms to better adapt wheat to heat stress.