Structural features of pentagonal Ag nanorods have been investigated by means of transmission electron microscopy (TEM). Cross-section observations directly reveal the remarkable fivefold twinning structure and related defects in this kind of nanomaterial. High-resolution TEM observations in combination with fast-Fourier processing indicate that the well-defined twinning relationship appears on at least two of the five twinning boundaries. Electron-energy-loss spectroscopy analysis on the center regions of the nanorods demonstrates that the Ag-M-4,M-5 peaks shift to lower energy in comparison with results from the Ag crystal.