Background and Objective: Cyclanthus bipartitus is an indoor ornamental plant. It’s fertilization relies on Cyclocephala sp. (Scarabaeidae). The absence of these pollinators could lead to lack of production of seed and therefore its vegetative propagation is obligatory. This study was carried out to determine the optimum concentration of benzylaminopurine (BAP), alpha-naphthalene acetic acid (NAA) and sucrose to be used in the Murashige and Skoogs (MS) media for in vitro propagation of Cyclanthus bipartitus and to evaluate the variation in DNA sequence of regenerated plants. Materials and Methods: Explants were cultured on sterilized MS medium containing different combinations of BAP, NAA and sucrose. The preparations were cultured for 10 weeks at temperature 25-28°C under 16 h of photoperiod with light intensity of 30 μmol m–2 sec–1 and the growth of the plantlets were monitored. Somaclonal variation amongst mother plants, daughter plants and the plantlets generated from the following three subculturing at the DNA level and was analyzed by using random amplified polymorphic DNA (RAPD). Data obtained were subjected to analysis of variance (ANOVA) using PROC GLM of SAS software. Results: Multiple adventitious shoots of Cyclanthus bipartitus were induced from lamina explants cultured on MS medium supplemented with various combinations of BAP, NAA and sucrose concentrations. The highest number of shoots, tallest shoots, highest number of roots and longest root were recorded in medium with a combination of 1.0 mg L–1 BAP, 0.5 mg L–1 and 30 g L–1 sucrose. Results of DNA analysis showed that 18 out of 26 score able bands were polymorphic and eight were monomorphic, which gave 69.2% polymorphism frequency. Conclusion: Optimum concentrations of BAP, NAA and sucrose for shoot and root development in vitro propagation of Cyclanthus bipartitus was 1.0 mg L–1 BAP, 0.5 mg L–1 NAA and 30 g L–1 of sucrose. 69.2% polymorphism was detected in regenerated plants indicating genetic variation had occurred amongst somaclones.