An efficient callus-mediated regeneration system was developed for high-frequency production of planting material of sugarcane genotypes LSC and B36464. Spindle leaf segments cultured on MS basal medium supplemented with 2,4-D or picloram at 1, 2, 3 or 4 mg/L resulted in callus induction. Callus induction was higher on 2,4-D amended medium compared to picloram. Nevertheless, for both auxins, callus induction improved significantly (p ≤ 0.05) with increasing concentration; the highest (82 and 82.5% for B36464 and LSC respectively) was achieved at 4 mg/L. For shoot induction, calli were transferred to MS medium supplemented with BAP (0.1, 0.5, 1.0 or 1.5 mg/L). The highest number of shoots (18.13 and 16.75 for B36464 and LSC respectively) was achieved at 1.5 mg/L. Serial subculture at four-week intervals on a higher concentration of BAP (2.5 mg/L), in combination with NAA (0.5 mg/L) and GA 3 (0.5 mg/L), resulted in a four-fold increase in shoot number within 16 weeks. On this medium, 40% of shoot clusters of B36464 formed well-defined shoots. On MS medium containing solely NAA (3 mg/L), 88 and 72% (B36464 and LSC respectively) formed roots. Post-flask acclimatisation of the plantlets led to 85 and 91% survival rates in LSC and B36464 respectively after which plantlets were successfully transferred to field conditions. The callus-mediated regeneration system reported in this study has the potential to sustainably provide sugarcane planting material for the emerging sugar industry in Ghana.