Polymer electrolyte fuel cells (PEFCs) used for marine vessels have been expected as an effective means to reduce the environmental burden caused by pollutant emissions. Since marine PEFCs operate under harsh conditions, such as ship vibrations and sea salt exposure, the diagnosis of abnormal operational conditions has a great importance when considering ways to improve the reliability and durability of marine PEFCs. Electrochemical impedance spectroscopy (EIS) is a technique suitable for real-time diagnosis of PEFCs in operation. In our experiment described in this paper, EIS was used to gauge the impact of factors that could determine the performance of PEFCs, namely operating temperatures, cathode fed air conditions of relative humidity (RH), flow rates and sea salt (NaCl) contamination. EIS spectra were obtained through Fast Fourier Transform (FFT) EIS techniques, and a transmission line model (TML), which included the ohmic resistance of the ionomer (Rion), was utilized as an equivalent circuit. Under conditions of dehydration (dry-up), we witnessed an increase in Rion, the ohmic resistance of the electrolyte film (Rohm) and the charge transfer resistance of the electrode (Rct). When liquid water was excessive (flooding), mass transfer resistance (Rmt) drastically increased in the cathode. All resistance values also rose after NaCl solution was injected into the cathode inlet gas. In particular, Rct and Rmt were the main factors that led to an increase in the polarization loss. Injecting distilled water after detecting NaCl contamination at an earlier stage is effective in mitigating irreversible degradation.