Technical Abstract: High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D). HRCT imaging is based on the same principles as medical CT systems, but a high intensity, focused synchrotron x-ray source results in decreased section thickness and image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source- Berkeley CA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem networks in excised samples and in living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods. Results demonstrate that HRCT can be applied to a range of plant species, including both herbaceous and woody, and plant organs (i.e. leafs, petioles, stems, trunks, roots). Figures presented within this paper help to demonstrate the range of vascular anatomy and the detail gathered from these scans for plant species ranging from redwood, walnut, oak, and maple tree saplings to sunflowers, grapevines, and ferns. Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (i.e. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT have provided promising new insights into plant vascular function.