Extreme weather events are expected to increase in frequency and intensity in South America, likely causing decreased plant productivity and altering species distributions. Yerba mate (Ilex paraguariensis) is a tree species native to South America and has an ecological, social, and economic importance in several countries. Natural forests and cultivated areas of yerba mate will be endangered by climate change because of the expected reduction in water availability. Here we determined how clonal genotypes of yerba mate (BRS BLD Yari, BRS BLD Aupaba, BRS 408, and EC40) respond to four levels of soil water holding capacity (100%, 80%, 60%, and 40% WHC) over 60 days, by evaluating the plants’ morphophysiological and biochemical characteristics. We observed a reduction in plant height and biomass accumulation related to the decrease in water availability; physiological and biochemical parameters indicated that water-deficit stress reduced the plants’ C assimilation and increased their production of bioactive compounds. BRS BLD Yari had a higher tolerance to low water availability, with greater biomass accumulation and photosynthetic rates that indicate greater water use efficiency. Understanding how different yerba mate genotypes respond to water deficit is essential for species conservation and developing climate-adapted breeding programs.