To better understand the startup process, the evolution of voltage output, energy gain and output, active biomass, and electrochemical performance of microbial fuel cells (MFCs) started up under different external resistances (10, 50, 250, and 1000 Omega) was investigated. In addition, the influences of external resistance and mass transfer on the anodic biofilm distribution of MFCs were also investigated. The results showed that MFCs with a decreased external resistance during startup had an increased evolution rate of energy gain, energy output, active biomass, and maximum power density but a reduced voltage evolution rate. A low external resistance for startup resulted in a thick and compact biofilm. As the flow field induced mass transfer, the biofilm was mainly distributed on the carbon cloth against the flow channel, and the biomass decreased along the flow direction. The compact biofilm on the development inside the electrode. surface resulted in a poor mass transfer and limited biofilm development inside the electrode.