In prospects of global climate change, heat stress is a rising constraint for the productivity of wheat (Triticum aestivum L.). It is a heat-susceptible crop beyond 17-23oC temperature throughout its phenological stages, flowering phase being the most sensitive stage. Chlorophyll a fluorescence parameter, maximum quantum yield efficiency of PSII (Fv/Fm) is used as a physiological marker for early stress detection in PSII in plants. We established a reproducible protocol to measure response of wheat genotypes to high temperature based on Fv/Fm. The heat treatment of 40°C in 300 µmol m-2s-1 PAR for 72h was appropriate to induce genotype dependent variation in Fv/Fm. This standardized protocol was used to phenotype wheat genotypes until the variation in the genotypes was consistently high with increased heritability for the trait, Fv/Fm. Mass screening of 1273 wheat genotypes in a milder stress of 38oC for 2h showed a heritability of 7%. However, a stronger stress at 40oC for 72h in repeated experiments on 138 extreme performing selected genotypes resulted in an increased genetic component of 15% which was further increased to 29% in the third selection cycle with 41 genotypes. Our protocol enabled the identification of contrasting wheat genotypes that can be used to study the genetic and physiological nature of heat stress tolerance to dissect quantitative traits into simpler and more heritable traits.