Wheat breeding nowadays must address producers and consumers' desire. According to the last FAO report, a dramatic decrease in wheat production is expected in the next decades mainly due to the upcoming climate change. The identification of the processes which are triggered by heat stress and how thermotolerance develops in wheat is an active research topic. Genomic approach may help wheat breeding since it allows direct study on the genotype and relationship with the phenotype. Here the isolation and characterization of four members of the chloroplast-localized small heat shock proteins (sHSP) encoded by the Hsp26 gene family is reported. Furthermore, two high throughput TILLING (Targeting Induced Local Lesions In Genomes) approaches in vivo and in silico were used for the identification of new alleles within this family. Small heat shock proteins are known to prevent the irreversible aggregation of misfolded proteins and contribute to the acquisition of thermotolerance. Chloroplast-localized sHSPs protect the photosynthetic machinery during episodes of high temperature stress. The modulation of the newly discovered genes within the sHsp26 family has been analyzed in vivo and by the ExpVIP platform widening the abiotic stress analysis; and their involvement in the heat stress response has been demonstrated. In addition, in this study a total of 50 TILLING mutant lines have been identified. A set of KASP (Kompetitive Allele Specific PCR) markers was also developed to follow the specific mutations in the ongoing backcrosses, applicable to high throughput genotyping approaches and usable in marker assisted selection breeding programs.