BackgroundCymbidium aloifoliumis a popular ornamental flower in Thailand with both economic and medical values. Polyploid induction techniques are used to improve plant quality. This study identified polyploidy levels ofC. aloifoliuminduction by colchicine. Protocorms ofC. aloifoliumwere treated on solid New Dogashima Medium (NDM) with various concentrations of colchicine (0, 0.01, 0.02, 0.03, 0.04 and 0.05% w/v) for 2, 4 and 8 weeks.ResultsColchicine effectively induced plant polyploidy. Tetraploid plants were found after treatment with 0.03% and 0.04% colchicine for 8 weeks, while at increased concentration and duration, survival, response and growth performance decreased. Tetraploid plants showed the lowest growth performance but highest size of stomatal and epidermal cells. Growth performance and leaf surface anatomy data were analyzed by Pearson’s correlation and PCA. Results showed that stomatal and epidermal cell sizes had strongly negative correlations with other variables, while HCA revealed that stomatal and epidermal cell sizes of tetraploid plantlets were larger but stomatal and epidermal cell densities decreased when compared with the diploids.ConclusionColchicine at suitable concentrations and duration produced polyploid plants with alteration of morphological and anatomical traits. This study provides potential information to support orchid quality production as ornamental plants and a source of pharmaceutical raw materials.