To understand the effect of substrate and temperature on methane production in high frigid wetlands, soil samples along the soil profile were collected from Zoige alpine wetlands, a “hotspot” of methane emission on Qinghai-Tibetan Plateau. Our result showed that temperature had a positive influence on methane production. However, acetate accumulation was negative related with temperature. We found that supplemental methanol and acetate strongly stimulated methane production. Such result indicated that methanol and acetate are major substrates for methane production in Zoige wetlands. Moreover, we also found that methane production rates decreased with soil depths due to the decrease of available substrate in deeper soil.