Copper Through-Silicon Via (TSV) is a key technology for three-dimensional integrated circuit applications [1], and its microstructure is being studied for a better understanding on its electrical and reliability characteristics. Observing the copper microstructure is crucial as it will provide information on how the copper TSV will behave under high temperature processes [2]. The shape of TSV itself will also affect the grain growth, and the resulting stress within the TSV as well as the surrounding silicon [3].Electron backscatter diffraction (EBSD) technique is commonly used for microstructure characterizations. For TSV, there have been studies on microstructure using twodimensional (2D) EBSD to obtain information such as grain size and orientation [4,5]. Our previous work showed that the grain size increases by about 20% in diameter after thermal annealing at 400°C, which indicated grain growth during the thermal process [5]. This paper will focus on the three dimensional (3D) EBSD characterization and analysis, and its advantages over 2D observation.