This study was designed to examine effects of high temperature, drought and exogenous abscisic acid (ABA) on membrane stability, osmotic adjustment, photosynthesis and the hormone status of two lucerne (alfalfa, Medicago sativa L.) genotypes contrasting in heat tolerance: Ameristand 801S (AS801) (heat-tolerant), and Aohan (heat-sensitive), The results showed that AS801 had lower electrolyte leakage, but higher chlorophyll content, net photosynthetic rate, stomatal conductance, proline content, ABA content and zeatin riboside (ZR) content than Aohan during 72 h of heat stress under well-watered conditions. Under drought conditions, however, only proline content and ZR content in roots, electrolyte leakage in leaves and roots, net photosynthetic rate, and ABA content were significantly different between the two genotypes. A foliar application of ABA to heat-stressed plants significantly decreased electrolyte leakage and stomatal conductance, and increased recovery in growth and leaf water potential in the two genotypes under both watering conditions. The other physiological responses measured differed under drought or well-watered conditions, and appeared to be genotype-specific. These results suggest that the physiological responses of heat-sensitive and heat-tolerant lucerne to heat stress under different soil-water conditions varied. The heat-induced changes in proline accumulation in roots, electrolyte leakage in leaves and roots, and photosynthetic rate could serve as early instant stress indicators for evaluating the tolerance of lucerne genotypes to heat stress under different soil water conditions.