Abstract Acacia mearnsii and Ilex paraguariensis are tree species of great social and economic importance in Brazil, demanding clonal cultivars. Their shoots possess a reservoir of totipotent cells with suitable potential for adventitious rooting, essential for mass production of high-quality seedlings. This study aimed to gain new insights into how anatomical barriers, i.e. sclerified tissues in the cortical region, may affect the adventitious rooting of cuttings from these species through histological examinations. For both species, histological analysis revealed significant diagnostic features. Tissue decay appears to be equivalent to an anatomical barrier in A. mearnsii. Starch abundance was notable in clones with higher rooting competence of I. paraguariensis, but they were not observed in the fundamental tissues of A. mearnsii, regardless of the rooting competence of the clone. The main differences in adventitious rooting were associated with the speed of response, initiated from cortical meristems, followed by differentiation of conductive tissue from newly formed tissue, connecting the periphery with the secondary vascular tissue. Thus, this newly formed tissue with parenchymatic structure provides the necessary structural basis for radial vascular connections. For both studied species, rhizogenesis presents distinct barriers to rooting, nevertheless these are not necessarily of anatomical nature.