Various factors were found to influence the in vitro induction and elongation of adventitious roots from walnut shoot microcuttings. Diverse walnut genotypes (Juglans regia, J. nigra × J. regia hybrids) and selected elite J. regia clones were micropropagated throughout the establishment of in vitro shoot-tip cultures. New evidence is presented here that demonstrates the importance of the genotype and juvenility of the plant material on the in vitro rooting ability. Selection of the best adapted genotypes to multiplication and rooting, and rejuvenation of mature clones through repetitive subcultures or micrografting were examined. Adult J. regia clones were rejuvenated through subsequent subcultures and their rooting was consequently improved. The same results were not accomplished by micrografting on juvenile shoots. A differential response to auxin type and concentration was observed for Juglans regia or J. nigra × J. regia clones. A short prerooting culture in multiplication medium, lowering the sucrose concentration in the root elongation medium and increasing the atmospheric carbon dioxide during the root elongation phase affected the number of shoots forming roots as well as the quality of plantlets and roots.