Vitis amurensis ‘Cheongsan’ is a plant with high economic value in both medical and agricultural applications. However, its utilization has been restricted owing to difficulties encountered when applying traditional mass propagation methods, requiring instead application of in vitro propagation methods for their mass scale production. Hence, this study was conducted to find the optimal plant growth regulators for shoot multiplication and root induction during in vitro propagation. Among the three cytokinins used at multiple concentrations for culture initiation and shoot multiplication, the most positive response was found with MS medium containing 5.0 μM 6-benzyladenine (BA), compared to more modest responses from other types of cytokinin, such as kinetin (KIN) and thidiazuron (TDZ). For root induction, medium supplemented with α-naphthaleneacetic acid (NAA) produced a callus and inhibited shoot growth in explants, whereas indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) did not create any significant problems, but did display differences in root induction efficiencies. Generally, root induction responses with IBA were better that those with IAA. The maximum rooting rates were observed without callus formation and no shoot growth inhibition from explants grown on media supplemented with 0.67 μM IBA. Further, inter-simple sequence repeat (ISSR) analyses revealed that micropropagated plantlets generated in medium supplemented with 5.0 μM BA and 0.67 μM IBA did not lead to genetic variation. Therefore, the application of the in vitro propagation method developed in this study could be used on a commercial scale and will offer opportunities to strengthen the industrial use of V. amurensis ‘Cheongsan’.