In vitro propagation of Paradox walnut rootstock (Juglans hindsii × J. regia) was investigated using apical and lateral meristems. A walnut-specific medium, DKW, was developed to support optimal multiple shoot development under 4.5 μM benzyladenine (BA) and 5 nm indolebutyric acid (IBA). The study tested various media (Cheng, B5, WPM, MS) for suitability in supporting multiple shoot formation. Nodal explants were surface-sterilized and cultured on media without hormones. The optimal medium for shoot formation was WPM with 4.5 μM BA and 5 nm IBA. The effects of IBA and naphthaleneacetic acid (NAA) on root induction were tested, with 30 μM IBA resulting in 70% rooting. The DKW medium, derived over 10 months, supported rapid shoot multiplication and minimal callus formation. Shoots were rooted and acclimated under mist conditions, with 5 mm IBA treatment leading to successful rooting within 10-14 days. Acclimation to field conditions was achieved after 6 weeks under mist and 4 weeks on a greenhouse bench. The DKW medium enabled the production of uniform rootstocks, overcoming limitations of traditional propagation methods. The study highlights the effectiveness of in vitro techniques for Paradox walnut propagation, supporting the development of a reliable method for vegetative propagation of superior clones.In vitro propagation of Paradox walnut rootstock (Juglans hindsii × J. regia) was investigated using apical and lateral meristems. A walnut-specific medium, DKW, was developed to support optimal multiple shoot development under 4.5 μM benzyladenine (BA) and 5 nm indolebutyric acid (IBA). The study tested various media (Cheng, B5, WPM, MS) for suitability in supporting multiple shoot formation. Nodal explants were surface-sterilized and cultured on media without hormones. The optimal medium for shoot formation was WPM with 4.5 μM BA and 5 nm IBA. The effects of IBA and naphthaleneacetic acid (NAA) on root induction were tested, with 30 μM IBA resulting in 70% rooting. The DKW medium, derived over 10 months, supported rapid shoot multiplication and minimal callus formation. Shoots were rooted and acclimated under mist conditions, with 5 mm IBA treatment leading to successful rooting within 10-14 days. Acclimation to field conditions was achieved after 6 weeks under mist and 4 weeks on a greenhouse bench. The DKW medium enabled the production of uniform rootstocks, overcoming limitations of traditional propagation methods. The study highlights the effectiveness of in vitro techniques for Paradox walnut propagation, supporting the development of a reliable method for vegetative propagation of superior clones.