Characterizing roots according to their branching order and anatomy is a useful approach for identifying functional differences within and among root systems. In this study, the root branching order and the anatomy of each root order (stele and cortex area) were examined in two-year-old “Duke 7” avocado (Persea americana Mill.) trees propagated by seed and by clonal techniques. The root systems were found to have three different root orders that exhibited differences in the occurrence of secondary xylem. Fine roots (first- and second-order roots) presented only primary growth, whereas the main roots (third-order roots) exhibited secondary growth. Transverse sections of roots from the different orders showed pentarch, hexarch or heptarch tracheal element distributions. Newly emerged, long, unbranched pioneer roots were observed only in the clonal trees and showed particular anatomical features, such as a larger diameter and a proportionally greater cortex area than other roots, as well as primary growth. Additionally, significant differences were found between clonal and seedling trees in the stele area of third-order roots; clonal propagation resulted in larger stele areas in this type of root. Our results suggest that propagation methods influence the presence of new pioneer roots and the anatomy of third-order roots; clonal root systems branch more extensively than seed-grown root systems and develop a vascular system with a larger transport capacity.