Progress in the study of stable isotope discrimination in carbon assimilation by aquatic macrophytes has been slower than for other groups of primary producers, such as phytoplankton and terrestrial plants. A probable reason has been the methodologies employed for such a study: field collections or long-term incubations, both relying on the observation of changes in carbon isotope composition of plant tissue. Here, we present a short-term incubation method based on the change in carbon stable isotope composition in water. Its fundamental advantage over the other approaches is that the change in stable isotope composition in water in a closed system is much faster than in the plant tissue. We applied the method to investigate the relationship between carbon assimilation intensity and isotope discrimination. The results included a relatively small discrimination in respiration, a significant influence of carbon assimilation rate on discrimination, and the suggestion of HCO3− or CO2 uptake in photosynthesis. The information gathered using this method would be difficult to obtain in other ways, and so we believe that it should contribute to a better understanding of the physiology and ecology of aquatic macrophytes.