Succulents are outstanding plants that have unique geometric leaf structures and have high adaptation ability in semi-arid environments which make them collectible plants to breeders and nursery growers. Among approximately 12,500 registered species of succulent plants, Echeveria is one of the most diverse genera belonging to the Crassulaceae family which exhibits diverse morphology with increasing horticultural demand to date. To improve the visual quality and produce new cultivars in a short time, chemical mutagenesis has been commonly applied to many ornamental plants. Because the outcomes of chemical mutagenesis depend on several factors (i.e., concentration, treatment duration, and plant characteristics), a research project was undergone that delved into the use of chemical mutagens (colchicine, oryzalin, ethyl methanesulfonate, methyl methanesulphonate, and sodium azide) at different concentrations and soaking time for 20 Echeveria cultivars, such as E. ‘Brave’, E. ‘Peerless’, E. ‘Viyant’, E. ‘Snow Bunny’, E. ‘Momotarou’, E. ‘Sistar’, E. ‘Doterang’, E. ‘Hyaliana’, E. ‘Cubic Frost’, E. ‘Benbadis’, E. ‘Tippy’, and E. ‘Dark ice’, from 2018 to 2021. Among the mentioned species, colchicine-treated E. ‘Peerless’ has been subjected to further study from its primary application (MV1) to the following generation (MV2) and conducted survival and mutant rate determination, phenotypic evaluation, ploidy level identification, stomata characteristics, and chromosome number counting of successfully mutated plants. Mutant plants treated with colchicine showed significant differences in terms of increased leaf thickness (0.51% - 2.61%), widened leaf apex (0.67% - 1.30%), and less prominent leaf tips compared to control plants for both MV1 and MV2. The color hue (a*) of MV1 plants and the color lightness (L*) of MV2 mutant plants were significantly affected by the colchicine treatment which indicated that mutants visually display darker leaf colors. Colchicine-treated E. ‘Peerless’ leaves had increased significantly in stomata size when concentrations were increased in terms of their stomatal length (0.90% - 1.33%) and stomatal pore size (0.68% - 1.15%) for both generations. Flow cytometry was conducted on MV1 plants which showed the diploid-tetraploid mixoploid peak for plants treated at 0.2% for 3 h, 0.6% for 3h, and 0.8% for 3 h while control plants had a diploid peak. Mitotic chromosome number counting of plants treated with 0.4% +12 h showed the doubling or increasing chromosome numbers in MV1 and MV2 (2n = > 200) compared to control plants (2n = >110) which confirms colchicine treatment affects ploidy levels. The result of this study suggests that the use of colchicine is an effective method to induce polyploidy in E. ‘Peerless’ creating novel genetic sources for developing and breeding new cultivars, which may be used in other related species.