Abstract In vitro rooting protocols were previously performed on Nothofagus nervosa to optimise the culture medium and environmental conditions. A qualitative and quantitative tissue differentiation study during adventitious rooting in N. nervosa under different light/dark conditions was carried out to increase knowledge of the rhizogenesis process in shoots. The histological events through the rooting period were consistent with the proposed biochemical markers (peroxidases, tissue nutrient contents, polyamines, flavonoids). Development of parenchymatic tissues, shown by the staining pattern of the cell contents, and cambium activity were the best tissues to define the rooting phases. Knowledge of the histological changes could be used to optimise a protocol for micro‐propagation improving timing, quantity, and quality of roots, which would positively affect the survival rate during hardening.