This study focused on understanding the impact of operating conditions on the temperature profile of the interface between the polymer electrolyte membrane (PEM) and the catalyst layer at the cathode (i.e., the reaction surface) in a single polymer electrolyte fuel cell (PEFC). A 1D multi-plate heat-transfer model based on the temperature data of the separator measured by the thermograph in a power-generation experiment was developed to evaluate the temperature profile of the reaction surface. The in-plane temperature distributions on the reaction surface, which was analyzed at twenty points were investigated in detail with the relative humidity, flow rate of the supply gas, and gas channel pitch of the separator as variables. The results showed that the temperature of the reaction surface increased with the gas channel pitch except when the flow rate and relative humidity of supply gas were low. The temperature of the reaction surface measured along the gas-flow direction from the inlet to the outlet of the cell by 1–2 K under all experimental conditions. The impact of the relative humidity of the supply gas on the temperature of the reaction surface was insignificant compared to that of the gas channel pitch. The results of this study suggest that the temperature of the supply gas should be set higher by 2 K and that the accumulated water should be removed from the turn-round part of the gas channel in order to realize an even in-plane temperature distribution on the reaction surface.