The quantum yield of photosynthetic electron transport (Phi PSII), evaluated by means of chlorophyll (Chl) fluorescence analysis, has proven to be a useful screening test for drought tolerance in durum wheat (Triticum durum Desf.). To explore the potential of this parameter further in detecting drought-tolerant genotypes, three cereal species were studied; Phi PSII measurements were carried out under two different gas mixtures, at three points of the induction curve (to obtain the maximal Phi PSII and both the transient and steady-state actual Phi PSII), and at three different water stress levels (moderate, severe and drastic). The species investigated were durum and bread wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.); two cultivars per species, characterized by different levels of drought tolerance, were tested. The two gas mixtures used were normal air (21% O-2, 0.035% CO2 in N-2) to monitor the whole photosynthetic process under physiological conditions, and CO2 enriched-low O-2 air (1% O-2, 5% CO2 in N-2) to monitor Phi PSII reduction under stress mainly related to Calvin cycle activity. When Phi PSII related to both assimilatory and non-assimilatory metabolism was evaluated, the cultivar differences observed under normal air were more representative of the agronomic performance upon drought stress than under high CO2-low O-2 air. Maximal Phi PSII showed no difference among either cultivars, gas mixtures or stress levels, the efficiency of excitation capture being highly resistant to drought. The Phi PSII evaluated during the transient yielded predictable values in respect of drought tolerance for durum wheat and barley cultivars, highlighting the key role of regulatory processes such as the Mehler peroxidase reaction and possibly also cyclic electron transport, in preventing overreduction under stress. The results clearly show that when Chi fluorescence analysis is used as a parameter in plant breeding, different experimental conditions should be used depending on the physiological mechanism that is bred or selected for.