The objective of this study was to investigate the optimal colchicine concentration and treatment time to induce polyploid in Doritis pulcherrima Lindl. Protocormswere treated with 0.05% and0.1% (w/v) colchicine in liquid NDM (New Dogashima Medium, 1993) for 24, 48 and 72 hours, and then transferred to NDM agar for 3 months. Chromosome numbers of D. pulcherrima were determined byusing aceto-orcien squash technique. The results showed that the chromosome number of non-treated D.pulcherrima was 2n = 2x = 38 while the treated ones with 0.05% and 0.1% colchicine for 72 hours were2n = 4x = 76. The surviving rate of plantlets decreased when colchicine concentration and treatment timeincreased. The optimal colchicine concentration was 0.05% and the optimal treatment time was 72 hours.Ploidy level of plant was also determined from leaves using flow cytometry.