ABSTRACT Sugarcane (Saccharum spp.) is an important economic crop that accounts for 70-80% of global sugar production. Nigerian domestic sugar production accounts for only 2.7% of the nation’s sugar consumption. Establishment of commercial sugarcane plantation requires a large number of planting material, which is difficult to obtain using the conventional method of propagation due to low multiplication rate and transmission of diseases from one generation to another. Tissue culture is a formidable tool that can be used for micro-propagation and genetic improvement of sugarcane. This work was carried out to develop a protocol for micropropagation of sugarcane using indirect organogenesis. Callus was established on Murashige and Skoog media fortified with 2, 4-Dichlorophenoxyacetic acid (2, 4-D) after incubation in the dark for four weeks. Shoot regeneration was achieved following culture of embryonic callus on regeneration media consisting of MS basal supplemented with 6-Benzylaminopurine (BAP) + Naphthalene acetic acid (NAA). Efficient regeneration of both embryogenic and non-embryogenic callus was achieved on media containing 2.5 mg/L 2, 4-D. Application of 2.5 mg/L or 3.0 mg/L BAP with 0.5 mg/L NAA significantly increased the regeneration capacity of the embryogenic callus. The Sugarcane varieties significantly varied in regeneration potential, with the highest regeneration recorded in M21-1988 and BD9800. The protocol could be adapted for the mass production of sugarcane planting materials and the subsequent establishment of a commercial plantation in Nigeria. Keywords: Indirect organogenesis, Sugarcane, 2, 4-D, Callus, in vitro, Regeneration