Waterlogging is a serious environmental stress on lucerne (Medicago sativa) affecting its agronomic performance. To facilitate the breeding process, efficient tools to screen a population of lucerne cultivars are needed. In this study, a comparative analysis of waterlogging effects on leaf photosynthesis, pigment composition, PSII photochemistry, and plant growth characteristics was undertaken using four different lucerne cultivars (Aurora, Hunter River, L153 and Sequel HR). Two-month-old plants, grown in half-strength Hoagland nutrient solution, were waterlogged for 16 days, and plant physiological characteristics were monitored at regular intervals (every few days). All cultivars had significantly reduced fresh and dry weight for both shoots and roots after 16 days of waterlogging. Root biomass showed a greater percentage of reduction than did shoot biomass. As waterlogging stress developed, chlorophyll content, CO2 assimilation rate, transpiration rate, stomatal conductance and maximal quantum efficiency of PSII (Fv/Fm) decreased significantly. Chlorophyll a and b content gradually decreased over the time of the experiment in the stressed cultivars, and leaf chlorosis became increasingly evident. Although most of the parameters showed significant changes as waterlogging progressed, limitations render some of them inapplicable for screening. It is concluded that for practical screening purposes, the Fv/Fm ratio is the most appropriate. A significant difference between control and waterlogged plants became evident as early as day 7. Possible physiological mechanisms involved are discussed.