Indulgence of heat defense mechanism is crucial to allay undesirable effects by developing significant heat tolerant plants. Translation of heat stress related genes into proteins is a key tolerance strategy tailored by plants. In order to understand the possible mechanisms of heat tolerance in wheat at proteomic level, two wheat genotypes (WH 730-heat tolerant; Raj 4014-heat intolerant) along with their 10 extreme recombinant inbred lines (RILs) were exposed to heat stress (35 °C for 6 h) to identify important stress related proteins. 2-DE coupled with MALDI TOF/TOF of wheat seedlings revealed 14 differentially regulated protein spots. Compared to Raj 4014, 3 proteins viz. Rubisco activase A, Con A and PEP carboxylase 1 were differentially regulated only in WH 730 implying their practical role in heat tolerance. Above and beyond, increased expression of cytochrome b6f complex and catalase in tolerant RIL population signifies their role in accelerated electron flow during heat stress to cope up with the stress. Our results suggests that, compared to intolerant parent and RILs, tolerant parent and RILs might be actively modulating protein involved in photosynthesis, signal transduction and defense which signifies the activation of adaptation mechanism under heat stress.