In this work, the influence of the single cell number on output performance, cell uniformity and thermal management of high temperature polymer electrolyte membrane fuel cells stack (HT-PEMFCs stack) was investigated by combining numerical simulation and experimental method. The numerical simulation results show that when the number of single cell of the stack increases from 10 to 60, the average single cell voltage decreases slightly from 0.6414 V to 0.6404 V, and the voltage range between single cells increases from 1.8 mV to 6.5 mV. The average working temperature between single cells increases from 431.01 K to 433.90 K, and the range of the working temperature of each single cell increases from 6.95 K to 10.22 K. The numerical simulation results indicate that with the increase of the number of single cells in the stack, the average single cell voltage of the stack has a slight downward trend, and the voltage range between the single cells has increased, the voltage consistency between the single cells has decreased. Furthermore, the temperature difference between the single cells has increased, the uniformity of the average temperature of the single cell itself has also decreased, and the difficulty of the thermal management of the stack has increased. Under the guidance of the simulation results, HT-PEMFCs stacks with 30, 60, and 120 single cells were assembled and evaluated. Under the operating condition of dry hydrogen/air gas and the discharge current of 33 A, the average single cell voltage of fuel cell stacks with 30, 60, and 120 single cells was 0.6566, 0.6548, and 0.6552 V, respectively. The single cell range increased from 24 mV to 59 mV, which showed good consistency with the simulation results and verified the effectiveness of the simulation results. Under the operating condition of dry hydrogen/air gas with the metering coefficient of 1.5/2.5, the fuel cell stacks show excellent output performance. The output power of the three stacks reaches 1.35, 2.64, and 5.28 kW at 80 A discharge current, respectively. The results of this work provide theoretical and practical guidance for the design, assembly and evaluation of kW-scale high-temperature polymer electrolyte membrane fuel cell stacks.