The deformability of red blood cells is an essential parameter that controls the rheology of blood as well as its circulation in the body. Characterizing the rigidity of the cells and their heterogeneity in a blood sample is thus a key point in the understanding of occlusive phenomena, particularly in the case of erythrocytic diseases in which healthy cells coexist with pathological cells. However, measuring intracellular rheology in small biological compartments requires the development of specific techniques. We propose a technique based on molecular rotors-viscosity-sensitive fluorescent probes-to evaluate the above key point. DASPI molecular rotor has been identified with spectral fluorescence properties decoupled from those of hemoglobin, the main component of the cytosol. After validation of the rotor as a viscosity probe in model fluids, we showed by confocal microscopy that, in addition to binding to the membrane, the rotor penetrates spontaneously and uniformly into red blood cells. Experiments conducted on temperature-stiffened red blood cells show that molecular rotors can probe their overall rigidity. A simple model allowed us to separate the contribution of the cytosol from that of the membrane, providing a quantification of cytosol rigidification with temperature, consistent with independent measurements of the viscosity of hemoglobin solutions. Our experiments demonstrate that the rotor can be used to quantify the intracellular rheology of red blood cells at the cellular level, as well as the heterogeneity of this stiffness in a blood sample. This technique opens up new possibilities for biomedical applications, diagnosis and disease monitoring.