A new approach, suitable for real-time implementation, is introduced for estimation of non-uniform internal temperature distribution in cylindrical lithium-ion cells. A radial 1-D model is used to estimate the distribution using two inputs: the real or imaginary part of the electrochemical impedance of the cell at a single frequency, and the surface temperature. The approach does not require knowledge of cell thermal properties, heat generation or thermal boundary conditions. The model is validated experimentally, the first time for such an approach, using a cylindrical 26650 cell fitted with an internal thermocouple. The cell is heated by applying (1) current pulses of up to ±20 A and (2) a 3500 s HEV drive cycle current profile, whilst monitoring the surface and core temperatures and measuring impedance at 215 Hz. During the drive cycle test, the battery core temperature increases by 20 °C and the surface temperature increases by 14 °C. The mean absolute error in the predicted maximum temperature throughout the cycle is 0.6 °C (3% of the total core temperature increase), in contrast to a mean absolute error of 2.6 °C if the temperature is assumed to be uniform (13% of the total core temperature increase).