In this paper, we describe the technique of on-axis transmission Kikuchi diffraction (TKD) in a scanning electron microscope and demonstrate its use in characterizing nanoscale crystal structures and defects in semiconductor materials and devices. We explain how we modified hardware and software to achieve an effective spatial resolution of 2 nm during orientation mapping without decreasing acquisition speed, indexing quality, and other performance parameters. The paper includes illustrations comparing sample-detector geometries for conventional EBSD, TKD, and on-axis TKD. It also presents examples of the types of images that can be obtained using on-axis TKD, including raw crystal orientation maps, diffraction patterns, pattern quality maps, time-resolved orientation maps showing microstructure evolution, and a sparse sample map showing the distribution of quantum dots on an electron transparent support film.