Abstract A protocol for in vitro polyploid induction using oryzalin was developed for Dendrobium , Epidendrum , Odontioda , and Phalaenopsis orchids (Orchidaceae). Protocorms and protocorm-like bodies (PLBs) in liquid nutrient media were subjected to oryzalin treatments of 14.4, 28.9, and 57.7 μM (w/v) concentrations for 3 and 6 days. Higher concentrations and longer treatment durations lowered the survival rates of the explants, but increased the number of polyploids produced. Stomatal guard cell lengths as measured with digital image analysis software, helped to identify several polypoids from digital images of leaf imprints. The optimal treatments were: 14.4 μM for 6 days in Dendrobium and Odontioda ; 57.7 μM for 6 days in Epidendrum ; and, 14.4 μM for 3 days in Phalaenopsis .