Methanol crossover largely affects the efficiency of power generation in the direct methanol fuel cell. As the methanol crosses over through the membrane, the methanol oxidizes at the cathode, resulting in low fuel utilization and in a serious overpotential loss. In this study, the commercial membrane electrode assemblies (MEAs) are investigated with different operating conditions such as membrane thickness, cell temperature, and methanol solution concentration. The effects of these parameters on methanol crossover and power density are studied. With the same membrane, increasing the cell temperature promotes the cell performance as expected, and the lower methanol concentration causes the concentration polarization effects, thus resulting in lower cell performance. Although higher methanol solution concentration can overcome the concentration polarization, a serious methanol crossover decreases the cell performance at high cell temperature. In this study, the open circuit voltage (OCV) is inversely proportional to methanol solution concentration, and is proportional to membrane thickness and cell temperature. Although increasing membrane thickness lowers the degree of methanol crossover, on the other hand, the ohmic resistance increases simultaneously. Therefore, the cell performance using Nafion 117 as membrane is lower than that of Nafion 112. In addition, the performance of the MEA made in our laboratory is higher than the commercial product, indicating the capability of manufacturing MEA is acceptable.