The distribution of ion conductive channels on the Nafion membrane surface, which determines the formation of the three-phase boundary, plays a very important role in improving the performance of proton-exchange membrane fuel cells. Therefore, understanding the microstructures at the catalyst layer/membrane interfaces of proton-exchange membranes is essential. Although current-sensing atomic force microscopy (AFM) can present some surface conductance data, localized impedance measurement providing more accurate proton-transport information is desirable. To obtain this information, in our study, localized electrochemical impedance spectroscopy was measured automatically with a home-built AFM-electrochemical impedance spectroscopy setup in which AFM was coupled with an impedance tester by a customized procedure. By this method, the localized proton-transport resistance at different humidities was observed in spatially diverse locations, and the value decreased as the membrane became hydrated. Furthermore, the microstructure of the Nafion membrane was numerically reconstructed at different hydration levels to examine the relationship between the membrane microstructural morphology and proton-transport resistance. The results showed that the spatial diversity of proton-transport resistance arose from the variable concentration of hydrophilic groups at the contact location of the AFM tip and the membrane, and from the heterogeneity of dry sulfonic acid groups in the membrane that creates local variation in water content.