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A considerable amount of water is stored in vegetation, especially in regions with high precipitation rates. Knowledge of the vegetation water status is essential to monitor changes in ecosystem health and to assess the vegetation influence on the water budget. In this study, we develop and validate an approach to estimate the gravimetric vegetation water content (mg), defined as the amount of water [kg] per wet biomass [kg], based on the attenuation of microwave radiation through vegetation. mg is expected to be more closely related to the actual water status of a plant than the area-based vegetation water content (VWC), which expresses the amount of water [kg] per unit area [m2]. We conducted the study at the field scale over an entire growth cycle of a winter wheat field. Tower-based L-band microwave measurements together with in situ measurements of vegetation properties (i.e., vegetation height, and mg for validation) were performed. The results indicated a strong agreement between the in situ measured and retrieved mg (R2 of 0.89), with mean and standard deviation (STD) values of 0.55 and 0.26 for the in situ measured mg and 0.57 and 0.19 for the retrieved mg, respectively. Phenological changes in crop water content were captured, with the highest values of mg obtained during the growth phase of the vegetation (i.e., when the water content of the plants and the biomass were increasing) and the lowest values when the vegetation turned fully senescent (i.e., when the water content of the plant was the lowest). Comparing in situ measured mg and VWC, we found their highest agreement with an R2 of 0.95 after flowering (i.e., when the vegetation started to lose water) and their main differences with an R2 of 0.21 during the vegetative growth of the wheat vegetation (i.e., where the mg was constant and VWC increased due to structural changes in vegetation). In addition, we performed a sensitivity analysis on the vegetation volume fraction (δ), an input parameter to the proposed approach which represents the volume percentage of solid plant material in air. This δ-parameter is shown to have a distinct impact on the thermal emission at L-band, but keeping δ constant during the growth cycle of the winter wheat appeared to be valid for these mg retrievals.