We examined the seasonal and inter-annual variation of leaf-level photosynthetic characteristics of three C4 perennial species, Cyperus articulatus, Panicum repens and Imperata cylindrica, and their response to environmental variables, to determine comparative physiological responses of plants representing particular microhabitats within a seasonal tropical floodplain in the Okavango River Delta, Botswana. Five measurement campaigns were carried out over a period of 2 y which covered two early rainy seasons, two late rainy seasons and one dry season. For all three species, light-saturated net photosynthetic rates (Asat) and stomatal conductance (gsat) decreased with decreasing soil water content with a seasonal range for Asat of approximately 5–45 μmol m−2 s−1, and for gsat of 0.03–0.35 mol m−2 s−1. The species representing the wettest microhabitat (Cyperus) had the highest gsat at low leaf-to-air vapour pressure deficits (Dl), the highest ratio of intercellular to ambient CO2 concentration (Ci/Ca), as well as the highest degree of variation in Ci/Ca from season to season. We interpret this as being indicative of its adaptation to a moist growth environment allowing for non-conservative water use strategies as soil moisture is usually abundant. For all three species there was significant variation in photosynthetic fluxes from one year to another that was related to variation in leaf nitrogen and phosphorus. This study shows that when assessing the role of savanna stands in large-scale carbon balance models, the remarkable inter-annual variation in leaf photosynthesis reported in this study should be taken into account.