Roots play an essential role in the acquisition of water and nutrients from soils in higher plants. Root anatomical traits have significant effects on root functions, including water transportation within the root system. Traits such as the size of xylem vessels influence axial water flow by controlling water conductance directly. Currently, two-dimensional microscopic images are often used to acquire root anatomical features; however, three-dimensional (3-D) anatomical analysis, which offers information of spatial distribution and connection relationships throughout root tissues, is hardly reported. We performed 3-D reconstruction and visualization of maize (Zea mays L.) root tissues based on X-ray micro-computed tomography and developed an image processing workflow for 3-D segmentation of metaxylem vessels. Three-dimensional quantitative analysis of metaxylem vessels from the first to the sixth whorl of maize crown roots was performed according to this procedure. The performance of this procedudure, based on accuracy validation, indicated that it was capable of making accurate analysis of root metaxylem vessel traits.