High-resolution X-ray micro-tomography (μCT) has emerged as one of the most promising new tools available to wood anatomists to study the three-dimensional organization of xylem networks. This non-destructive method faithfully reproduces the spatial relationships between the different cell types and allows the user to explore wood anatomy in new and innovative ways. With μCT imaging, the sample can be visualized in any plane and is not limited to a single section or exposed plane. Conventional CT software aids in the visualization of wood structures, and newly developed custom software can be used to rapidly automate the data extraction process, thereby accelerating the rate at which samples can be analyzed for research. In this review the origins of xylem reconstructions using traditional methods are discussed, as well as the current applications of μCT in plant biology and an overview of pertinent technical considerations associated with this technique. μCT imaging offers a new perspective on wood anatomy and highlights the importance of the relationships between wood structure and function.