Water molecules play an important role in the structure, function, and dynamics of (bio‐) materials. A direct access to the number of water molecules in nanoscopic volumes can thus give new molecular insights into materials and allow for fine‐tuning their properties in sophisticated applications. The determination of the local water content has become possible by the finding that H ₂ O quenches the fluorescence of red‐emitting dyes. Since deuterated water, D ₂ O, does not induce significant fluorescence quenching, fluorescence lifetime measurements performed in different H ₂ O/D ₂ O‐ratios yield the local water concentration. We combined this effect with the recently developed fluorescence lifetime single molecule localization microscopy imaging (FL‐SMLM) in order to nanoscopically determine the local water content in microgels, i.e. soft hydrogel particles consisting of a cross‐linked polymer swollen in water. The change in water content of thermo‐responsive microgels when changing from their swollen state at room temperature to a collapsed state at elevated temperature could be analyzed. A clear decrease in water content was found that was, to our surprise, rather uniform throughout the entire microgel volume. Only a slightly higher water content around the dye was found in the periphery with respect to the center of the swollen microgels.