Abstract The transmission electron microscope (TEM) is a powerful imaging, diffraction and spectroscopy tool that has revolutionized the field of microscopy. It has contributed to numerous breakthroughs in various scientific disciplines. TEM-based techniques can offer atomic resolution as well as elemental analysis, which benefit the study of epitaxial semiconductors and their related optoelectronic devices on the atomic scale. The design and optimization of the device performance depend on three key factors: the control of strain at nanometer scale, control of the formation and propagation of defects as well as the control of local electronic properties. Manipulation and optimization are only possible if the key factors can be characterized precisely. Herein, the TEM techniques for strain analysis, defect characterization and bandgap evaluation are reviewed and discussed. Lately, with the development of in-situ TEM techniques, researchers have been able to observe dynamic processes and study the behaviour of materials and devices under realistic conditions (in gaseous atmosphere or in liquids, at elevated or cryogenic temperatures, under strain, bias or illumination) in real-time with extremely high spatial resolution. This review explores the impact and significance of in-situ TEM in the field of semiconductors.