As an assessment of substantial equivalence and environmental risks of branchless chrysanthemum GM lines (LeLS69, LeLS76, LeLS80) developed in Korea, we investigated their degree of branchlessness and developmental characteristics including growth and flowering, as well as the stability of populations of insect pests and soil microbes. The GM lines were compared with a control variety, 'Jinba'. LeLS80 showed the highest level of branchlessness among the three GM lines, whereas abnormal axillary buds developed in lines LeLS76 and LeLS69. There were no significant differences between the growth characteristics of 'Jinba' and the GM lines. The branchlessness LeLS80 was increased during the summer season, suggesting that it is a useful germplasm for developing elite branchless cultivars. These GM lines showed no significant differences in the occurrence of five insect pests including aphids, mites, leaf miners, thrips, and whiteflies. Similarly, for soil microbes including bacteria and actinomycetes, there were no significant differences between 'Jinba' and the GM lines. However, there was a higher occurrence of fungi in the GM lines compared to 'Jinba'. Our findings suggest that 'Jinba' and the GM lines are substantially equivalent, although further monitoring studies are needed to confirm this.