The in-cylinder temperature information is critical in the field of auto-ignition control in advanced combustion modes. However, the in-cylinder temperature is hard to be directly measured at low cost in production engines. In this paper, a cycle-by-cycle estimation method is proposed for the in-cylinder temperature at the crank angle of intake valve closing (IVC), referred to as Tivc. Through investigating the thermodynamics of Tivc, an Extended Kalman Filter (EKF) based method was devised by utilizing the measurable temperature information from the intake and exhaust manifolds. The proposed method was validated through high-fidelity GT-Power engine model simulation.