This study explores the in-vitro regeneration of sugarcane (Saccharum officinarum L.) genotypes in Bangladesh, aiming to optimize micropropagation protocols that can enhance sugarcane productivity and genetic diversity. The research evaluates the effects of various concentrations of auxins and cytokinins on callus induction, shoot regeneration and rooting in three sugarcane varieties: Isd 37, Isd 40 and ZH 110-65. The results demonstrated that 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) was the most effective for callus induction, yielding a high initiation rate of 97.33 % in Isd 40. For shoot regeneration, the optimal concentration of 1.0 mg/L Benzylaminopurine (BA) produced the highest number of shoots per culture, reaching 17.0 shoots per culture in Isd 40. Rooting was most successful with 5.0 mg/L Naphthalene acetic acid (NAA) which resulted in an average of 9.36 roots per shoot in Isd 40. Acclimatization trials showed that 65-80 % of the rooted plantlets survived under ex vitro conditions. This research provides a robust, reproducible tissue culture protocol for sugarcane that can be utilized to propagate disease-free, high-yielding and genetically diverse genotypes. The findings hold significant potential for improving sugarcane breeding and commercial production in Bangladesh, addressing the growing demand for high-quality sugarcane and supporting the sustainable growth of the local industry. The study lays a foundation for future crop improvement programs and germplasm conservation.