The establishment of an efficient Agrobacterium–mediated transformation protocol for a recalcitrant crop like high oleic sunflower genotypes, cv. Capella and SWSR2 inbred line, is presented in this paper. The protocol requires the identification and optimization of parameters affecting T-DNA delivery and plant regeneration. The basis for identifying the most appropriate conditions for transformation was the fluorometric, histochemical GUS activity coupled with plant cell vitality. Agrobacterium tumefaciens–mediated transformation was performed using strain LBA4404 for cv. Capella and strain GV3101 for the inbred line. Both strains harboring the pBI121 plasmid with the gus gene were under the control of 35S promoter. The flourometric GUS activity was increased in cv. Capella and SWSR2 inbred line 1.9 and 1.6 fold, respectively, relative to transformation without inducers, by optimizing the bacterial density to OD600=1.0, by splitting shoot apices and by using MS medium as a co-cultivation medium supplemented with 200 µM acetosyrin gone. Pre-culturing the explants for three days on SIM2 medium before the bacterial inoculation followed by co-cultivation for 72 h increased the percentage of the GUS expression to 40% and 30% in cv. Capella and SWSR2 inbred line, respectively. It also improved the regeneration percentage as well as the vitality of cells.