Abstract New ornamental varieties with higher quality can be created via artificial polyploid induction. In the present study, the first polyploid induction of Paphiopedilum villosum was described. In vitro young shoots of Paphiopedilum villosum were produced shoots to be used as material for inducing autopolyploidization. Initial shoots were extended in the dark to form three distinct stem nodes and then adventitious shoots were induced from nodal explants from the elongated shoots. Therein, the effects of cytokinins (BA, KIN, and TDZ) and node position on shoot induction were investigated. The shoots (1.5 cm high) derived from the first nodes in 0.5 mg/L TDZ supplementation medium were incubated in colchicine solution at different concentrations and durations and then transferred to rooting medium. Polyploid induction rate reached 19.88% in the treatment of 50 μM colchicine and 6 days. Among obtained polyploidy plantlets, tetraploids (88.24%) and mixoploids were identified by flow cytometric analysis and chromosome counts of root tip tissue squashes. The combination of shoot regeneration from stem nodes and colchicine treatment gives an effective method for the production of polyploid plantlets for further breeding purposes of P. villosum as well as other endangered orchids.