Perfluorosulfonic acid ionomers (PFSI) with different side-chain lengths have been investigated with respect to their morphology and electrochemical properties in vanadium flow batteries (VFB). The results indicated that the membrane with the shortest side chains (SSC-M2) displayed small ion clusters and a low degree of hydrophobic–hydrophilic separation, which is favourable to reduce the cross-over of vanadium ions in the VFB. SSC-M2 shows a similar proton conductivity to Nafion, which carries longer ionic side chains but with much lower ion permeability. As a result, the VFB assembled with SSC-M2 exhibited a superior coulombic efficiency and a voltage efficiency close to that of Nafion115. In situ mass transfer revealed that SSC-M2 had a remarkably low degree of vanadium and water transfer across the membrane, which resulted in lower capacity fading than in the case of Nafion115. These results indicate that a membrane with short side chains is an ideal option in the fabrication of high-performance VFBs with low capacity loss.