Properties of the microvasculature that contribute to tissue perfusion can be assessed using immunohistochemistry on 2D histology sections. However, the vasculature is inherently 3D and the ability to measure and visualize the vessel wall components in 3D will aid in detecting focal pathologies. Our objectives were (1) to develop a method for 3D measurement and visualization of microvasculature in 3D, (2) to compare the normal and regenerated post-ischemia mouse hind limb microvasculature, and (3) to compare the 2D and 3D vessel morphology measures. Vessels were stained for smooth muscle using 3,3'-Diaminobenzidine (DAB) immunostain for both normal (n = 6 mice) and regenerated vasculature (n = 5 mice). 2D vessel segmentations were reconstructed into 3D using landmark based registration. No substantial bias was found in the 2D measurements relative to 3D, but larger differences were observed for individual vessels oriented non-orthogonally to the plane of sectioning. A larger value of area, perimeter, and vessel wall thickness was found in the normal vasculature as compared to the regenerated vasculature, for both the 2D and 3D measurements (p < 0.01). Aggregated 2D measurements are sufficient for identifying morphological differences between groups of mice; however, one must interpret individual 2D measurements with caution if the vessel centerline direction is unknown. Visualization of 3D measurements permits the detection of localized vessel morphology aberrations that are not revealed by 2D measurements. With vascular measure visualization methodologies in 3D, we are now capable of locating focal pathologies on a whole slide level.