High-temperature stress affects all growth stages of crops and ultimately yields. This is further aggravated by other environmental stresses like intermittent drought and high light. Management options are few and hence developing intrinsically tolerant plants is essential to combat the situation. As thermotolerance is a multigenic trait, emphasis needs to be on relevant approaches to assess genetic variability in basal and acquired tolerance. This is in fact the major aspect in crop improvement programmes. The relevance of temperature induction (acclimation) response (TIR), a high throughput approach to identify thermotolerant individuals and its utility as potential screening method is described here. This is based on the concept that stress-responsive genes are expressed only during initial stages of stress (acclimation stress) and bring about requisite changes in cell metabolism for adaptation. The fact that acclimation response is ubiquitous has been demonstrated in different crop plants in our studies and by others. Significance of acclimation in acquired tolerance and thus in assessing genetic variability in thermotolerance is discussed. The limitations of present approaches to validate the relevance of specific stress genes either in transgenics or in mutants or knock downs have been analyzed and the need to characterize transformants under conditions that trigger acquired tolerance is also highlighted. This review also focuses on the potential of exploiting acclimation response approach to improve the thermotolerance of crop plants by suitable breeding strategies.