We report on a newly-developed membrane stiffness-based separation of vesicles using a thermally-assisted acoustophoretic approach. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to the change in the acoustic compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature, corresponding to the temperature at which the acoustic contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop a separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. Our studies suggest that this method may be applied for the separation of cells affected by diseases that affect the cellular stiffness.