Drought stress is one of the main environmental factors limiting plant photosynthesis. Due to climate change drought problems in the world's agriculture will increase. Alfalfa, drought-tolerant crop, generally tolerates water shortages quite successfully, but its yield may fluctuate appreciably under drought conditions. Therefore, strategies for sustainable use of water and drought resistance improvement based on the physiological traits are urgent and physiological approaches should be integrated in conventional breeding. The first condition for a successful introduction of physiological traits in a breeding program is to determine the degree of genetic variability for the trait (traits) of interest. Research was performed with 24 alfalfa genotypes under vegetation house conditions at two water status regimes. The objective was to identify the available genetic variation and to establish efficient testing methods for characters which might positively influence alfalfa performance under drought conditions. Among the many characters that might be important for alfalfa performance under water stress, our research focused on stomatal resistance and photochemical PSII chlorophyll fluorescence parameter (Fv/Fm, index of performance). The analysis of variance regarding stomatal resistance showed a very significant influence of the treatment, genotype and their interaction. The variance of treatment was higher than the variance due to genotypes. The quantum of photosynthetic yield efficiency (Fv/Fm) was not significantly influenced by drought stress but the index of performance increased very significantly in drought stressed plants, compared to the control. There were significant correlations between stomatal resistance and biomass yield and also between stomatal resistance and drought susceptibility index, which opens new opportunities for improving drought resistance in alfalfa. We suggest that for improving resistance of alfalfa synthetic cultivars to drought, each of the genotypes components must have at least some of the traits involved in drought resistance.