Diagnosis of stack performance is of importance to DMFC research. Electrochemical impedance spectroscopy was used as a diagnostic tool for direct methanol fuel cell stack,capable of separating individual contribution to the overall polarization of fuel cells under load .This paper presents the diagnostic testing results of a six-cell DMFC stack with an active area of 48.5 cm2 by electrochemical impedance spectroscopy (EIS). By using this method, the process taking place in DMFC are characterized under realistic operating condition the effects of temperature, flow rate, and methanol concentration on the stack impedance spectra were examined. The results of the electrochemical impedance analysis show that with increasing temperature, the charge transfer resistance decreases due to the slow oxygen reduction reaction (ORR) process at low temperature. If the stack is operated at a fixed air flow rate, a low frequency arc appears and grows with increasing current due to the shortage of air. the ability of EIS to reveal these phenomena proved to be highly useful in the identification of performance issues in individual cells of a six-cell DMFC stack