<p>The Purple Gia Hac orchid (<i>Dendrobium anosmum</i>) is one of the most popular orchid species in Vietnam and has been excessively exploited due to its high aesthetic value. Therefore, genetic conservation and improvement efforts are essential for the preservation and development of this species. In plant breeding, colchicine-based polyploidization offers advantages over traditional hybridization by overcoming infertility and incompatibility barriers. In this study, we established the first protocol for polyploidization of <i>D. anosmum</i> callus, regeneration of in vitro plantlets from colchicine-treated calli, and acclimatization of micropropagated plantlets. Immersion of calli in 50 mg/L colchicine for two hours was optimal, yielding calli with the highest proportion of mutant phenotypes. MS medium supplemented with 1.5 mg/L 6-benzylaminopurine and 0.5 mg/L α-naphthaleneacetic acid was optimal for shoot regeneration, producing maximum mean shoot height (2.34 cm) and leaf number per shoot (3.33) after eight weeks of culture. The exhibition of enlarged morphological characteristics, including thickened and swollen stems and leaves, was found in shoots regenerated from colchicine-treated calli. Microscopic anatomical comparisons revealed that colchicine-treated plantlets/shoots had enlarged leaf epidermal cells, stomata, nuclei, and root hairs relative to untreated controls. Candidate polyploid lines also showed alterations in pigment contents and produced flowers with more intense purple coloration and color patches. Karyotyping confirmed high proportions of polyploid cells (66.19–84.48%) in candidate lines. These results demonstrate that colchicine-induced polyploidization holds promise for the aesthetic improvement of <i>D. anosmum</i> through subsequent selection of stable polyploid lines.</p>