Twelve accessions including two diploid, four tetraploid and six hexaploid genotypes were used in this study to assess heat tolerance in wheat. All possible crosses were made and the resulted hybrids with their parents were subsequently evaluated for heat tolerance in two sowing dates. Evaluation was based on 1000 grain weight, grain yield per plant, cell membrane thermostability and tetrazolium chloride reduction. Results revealed significant variability among wheat genotypes and hybrids in all evaluated traits under favorable and heat stress conditions. However, tetraploid genotypes showed the highest performance under heat stress followed by hexaploid and diploid genotypes, respectively. According to results of heat tolerance index, one tolerant and one susceptible parent from each ploidy level along with 6 related interspecific hybrids were chosen to be involved in molecular analysis. Target region amplification polymorphism (TRAP) markers were used based on fixed primers of heat shock protein genes. TRAP clearly confirmed the morpho-physiological findings and generated a dendrogram which was quietly like that of morpho-physiological data. TRAP was able to generate one and five specific bands for diploid and hexaploid genotypes respectively, while no specific bands were generated for tetraploid. In addition, the different genomes showed some shared bands between each other revealing their relationship. Interestingly, two specific bands for tolerant genotypes of different ploidy level were generated which were absent in all susceptible genotypes. Findings herein are of high importance and could help in successive breeding programs for wheat improvement.