To investigate the performance degradation characteristics of direct methanol fuel cell (DMFC) under two different operating conditions, namely, the worldwide harmonized light vehicle test cycle (WLTC) and the Chinese light vehicle test cycle (CLTC), we adopt a combination of polarization curves, equivalent circuit models, and the distribution of relaxation times (DRT) to analyze the performance degradation characteristics of DMFC. The changes in each polarization process of DMFC are explained by calculating the DRT from EIS according to the evolution of the waveforms. The results show that the degradation of WLTC condition is greater than that of CLTC condition. Under both operating conditions, the obstruction of the mass transfer process hindrance plays a leading role in the performance degradation of DMFC; the rate of change of the mass transfer process hindrance is 2.39 m Ω/h under the WLTC operating conditions and 0.764 m Ω/h Under the CLTC operating conditions; the oxygen reduction reaction hindrance is unaffected by the operating conditions. Under CLTC operating conditions, its significant dynamic fluctuations and abundant transient operating conditions pose a great obstacle to proton transport, thereby effectively reducing the membrane-bound water content. In comparison, it has less impact on the mass transfer obstruction, and thus promotes an increase in the rate of oxygen diffusion. A fuel cell degradation model is built to characterize the ageing state of DMFCs based on the distribution of relaxation times hindered by the oxygen reduction reaction. It provides a reference for the health state assessment and optimization in DMFC operation.