Alfalfa (Medicago sativa L.) is a valuable forage legume, but its production is largely affected by high temperature. In this study, we investigated the effect of heat stress on 15 alfalfa cultivars to identify heat-tolerant and -sensitive cultivars. Seedlings were exposed to 38/35 °C day/night temperature for 7 days and various parameters were measured. Heat stress significantly reduced the biomass, relative water content (RWC), chlorophyll content, and increased the electrolyte leakage (EL) and malondialdehyde (MDA) content of heat-sensitive alfalfa cultivars. However, heat-tolerant cultivars showed higher soluble sugar (SS) and soluble protein (SP) content. The heat tolerance of each cultivar was comprehensively evaluated based on membership function value. Cultivars with higher mean membership function value of 0.86 (Bara310SC) and 0.80 (Magna995) were heat tolerant, and Gibraltar and WL712 with lower membership function value (0.24) were heat sensitive. The heat tolerance of the above four cultivars were further evaluated by chlorophyll a fluorescence analysis. Heat stress significantly affected the photosynthetic activity of heat-sensitive cultivars. The overall results indicate that Bara310SC and WL712 are heat-tolerant and heat-sensitive cultivars, respectively. This study provides basic information for understanding the effect of heat stress on growth and productivity of alfalfa.