In view of the global climate change, heat stress is an increasing constraint for the productivity of wheat (Triticum aestivum L.). Our aim was to identify contrasting cultivars in terms of heat tolerance by mass screening of 1274 wheat cultivars of diverse origin, based on a physiological trait, the maximum quantum efficiency of PSII (Fv/Fm). A chlorophyll fluorescence protocol was standardised and used for repeated screening with increased selection pressure with a view to identifying a set of cultivars extreme for the trait. An initial mass screening of 1274 wheat cultivars with a milder heat stress of 38°C in 300µmolm-2s-1 for 2h with preheating at 33-35°C for 19h in 7-14µmolm-2s-1 light showed a genetic determination of 8.5±2.7%. A heat treatment of 40°C in 300µmolm-2s-1 for 72h in the second screening with 138 selected cultivars resulted in larger differentiation of cultivars with an increased genetic component (15.4±3.6%), which was further increased to 27.9±6.8% in the third screening with 41 contrasting cultivars. This contrasting set of cultivars was then used to compare the ability of chlorophyll fluorescence parameters to detect genetic difference in heat tolerance. The identification of a set of wheat cultivars contrasting for their inherent photochemical efficiency may aid future studies to understand the genetic and physiological nature of heat stress tolerance in order to dissect quantitative traits into simpler genetic factors.