Abstract: To explore the mechanism and provide a theoretical basis for the development and utilization of color mutants, Cymbidium tortisepalum Fukuy. var. longibracteatum with verge line pattern leaves and its parent leaves were used to investigate the characteristics of leaf variegation by paraffin section, scanning electron microscopy, and transmission electron microscopy. Results showed significant differences in the anatomical structure between the mutant and its parent. The epidermal thickness and mesophyll cell area of the mutant leaves were larger than those of the parent, whereas the number of vessels was lower. Scanning electron microscopy showed obvious differences in the micro-characteristics of the leaf surface between the mutant and parent. Stomatal area and density were smaller in the mutant leaves than that of the parent; furthermore, stomatal closure was higher in the mutant leaves than in the parent, with a frequency of 56% of the total number of stomata. Transmission electron microscopy showed no grana lamellae or starch grains, but many osmiophilic droplets in the mutant leaves, and the membrane of the chloroplast was disjointed. These results showed significant differences in the leaf structure between the mutant induced by tissue culture and its parent.