The optimization of direct and indirect in vitro explants regeneration represents one of the most important bottlenecks for sugarcane genetic transformation by Agrobacterium tumefaciens. Thus, this study aimed to assess the direct and indirect regeneration capacity of 11 sugarcane cultivars belonging to the Programa Cana IAC, in order to identify those more suitable for Agrobacterium transformation assays. All the genotypes that produced embryogenic calli were subjected to infection with Agrobacterium tumefaciens under different conditions. The explants were inoculated with the strains C58C1 and EHA-105 carrying the superbinary vectors pSoup and pAL156, which contains the bar (pat) and GUS (uidA) genes, in three different optical densities (OD600 0.4, 0.6, 0.8). Analysis of the expression of GUS reporter gene was performed with three selected genotypes. The highest rates of direct (~ 75%) and indirect (~ 70%) regeneration were observed for the cultivar IACSP96-5000. This cultivar also had the best rates for indirect regeneration when embryogenic calli were subjected to infection by Agrobacterium. GUS-positive calli, at a rate of 8.33%, were obtained only for the genotype IACSP95-5000 inoculated with OD600 = 0.8. Moreover, the Agrobacterium strains used did not significantly influence the regeneration capacity of explants. But the strong dependence on the sugarcane genotype was statistically evident in all experiments. Thus, the OD's, the origin of the explants, and especially the plant genotype seem to be the factors determining the success of this methodology in sugarcane genetic transformation.