The study aimed to work out a protocol for callus initiation from five genotypes of Cyamopsis tetragonoloba L. plant (Gm2, Gm5, Gm6, Gm8 and Gm34). Murashige and Skoog (MS) media supplemented with 3% sucrose were used as media for culture initiation. The MS medium was supplemented with different combinations of growth regulators (BAP, IBA, NAA and 2,4-D) at one concentration. Three type of explant used were; radicle, hypocotyl and cotyledonary leaf. Explants of five genotypes of C. tetragonoloba were cultured in Murashige and Skoog media supplemented with different growth regulators. Results show that M2 (with NAA) failed to form callus for the five genotypes, while M1 (with IBA) and M3 (with 2-4, D) initiated callus for the five genotypes. M3 was better than M1 for callus formation for the C. tetragonoloba genotypes. It was concluded that the five genotypes of C. tetragonoloba have responded differently to the plant growth regulators.