Micropropagation techniques allow producing large numbers of clones of genetically identical plants. However, there is evidence of disorders in internal structures due to sophisticated in vitro conditions. Such variations are responsible for the mortality of plantlets in the field and cause huge loss to the tissue culture industry. Anatomical evaluation at different growth conditions allows for understanding structural repair of in vitro raised plantlets. Therefore, the present study was aimed to identify the structural changes that occurred in micropropagated plants of Vitex negundo under heterotrophic, photomixotrophic, and photoautotrophic conditions. To achieve this, structural variations were analyzed in the plantlets obtained from in vitro, greenhouse and field transferred stages using light microscopy. Underdeveloped dermal tissues, palisade cells, intercellular spaces, mechanical tissues, vascular bundles, and ground tissues were observed with the plants growing under in vitro conditions. The self-repairing of structural disorders and transitions in vegetative anatomy was observed during hardening under the greenhouse environment. Field transferred plantlets were characterized by well-developed internal anatomy. These findings showed that the micropropagated plantlets of V. negundo were well-adapted through a series of self-repairing the in vitro induced structural abnormalities at the subsequent stages of plant development.