The uncontrolled deposition/dissolution process of lithium dendrites during electrochemical cycling in batteries limits the large-scale application of Li metal anodes. Investigating the microstructure of Li dendrites is a focal point. Currently, the only way to protect and observe sensitive Li dendrites is through low-temperature transmission electron microscopy (LT-TEM), whereas room-temperature characterization is still lacking. In this work, the room-temperature microstructure of Li dendrites was obtained by TEM using both vacuum- and inert-gas-transfer methods. Detailed comparison between LT- and room-temperature (RT-)TEM characterizations was provided to show the pros and cons of each method. Especially, RT-TEM shows the advantage of flexible incorporation with multifunctional characterizations, such as 3D tomography. By using RT-TEM, microstructural evolution of Li dendrites during the electrodeposition/dissolution process, including increase of the quantity of inorganic Li2O compounds in the solid electrolyte interphase, lateral growth behavior, and two types of inactive Li, has been revealed, enriching the understanding of the structure-property relationship of Li dendrites.