Wheat (Triticum aestivum L.) is a heat-susceptible crop throughout its phenological stages, flowering phase being the most sensitive stage. Early stress detection method with advanced physiological measurements may provide new dimensions to establish a high throughput phenotyping technique. Chlorophyll a fluorescence has been a versatile tool in photosynthesis research to measure plant responses to various abiotic stresses that affect PSII. We aim to establish a reproducible protocol to measure response of wheat genotypes to high temperature, based on the physiological marker, maximum quantum yield efficiency of PSII photochemistry (Fv/Fm). We subsequently used this standardized protocol for mass screening of wheat genotypes. Our results showed that the temperature of 40°C in 300 µmol m-2s-1 light for 72 h was appropriate to induce heat stress to reveal genetic variation among genotypes. Initial phenotyping of 1300 wheat genotypes in a milder stress at 38oC for 2 h showed a heritability of 7% for Fv/Fm. However, a stronger stress at 40oC for 72 h in repeated experiments on 138 extreme performing lines resulted in a genotype dependent drop in Fv/Fm and an increased genetic component of 15%. Our protocol seems to be stable over environments since interaction between genotypes and the three repeated experiments separated in time was not statistically significant. The chlorophyll a fluorescence protocol may enable identification of wheat lines reliably more or less tolerant to heat stress. Such differential lines can subsequently be used to study the genetic and physiological nature of stress tolerance, facilitating genetic dissection of quantitative trait into simpler and more heritable traits.