Heat stress alters various physiological phenomenons which adversely affect yield components and ultimately lead to a severe reduction in economic yield in wheat (Triticum aestivum L.). To cope with heat stress, plants have army of heat shock proteins (HSPs) which acts as molecular chaperones stabilizing the polypeptides and membranes. The thermo-tolerance ability of different genotypes of a plant species resides in their genetic diversity of HSP genes. In this study, association of HSPs with physiological and yield traits under heat stress was investigated. Results showed that wheat genotypes differed significantly in their response to high temperature. Single-strand conformation polymorphism (SSCP) analysis of targeted coding sequence of different HSP genes produced seven different haplotypes from HSP 16.9, while only two haplotypes were produced from HSP 23.5, HSP 90a and HSP 90b. Among 25 SSCP variants detected in HSP 16.9 targeted coding sequence, 12 were polymorphic and three of them were found significantly associated with canopy temperature (CT), relative water content (RWC), thousand grain weight (TGW) and normalized difference vegetation index (NDVI). These associated alleles explained 11.4 to 32.9% of the variation for individual trait. The association between HSP variants and these traits may provide new insight for HSPs potential contribution to thermo-tolerance which can be used for improvement of thermo-tolerance in wheat through marker assisted selection.