An efficient and reproducible Agrobacterium tumefaciens-mediated transformation method was developed for Gossypium hirsutum L. ‘KC3’ using a shoot apex explant with microinjection and sonication. To optimize transformation efficiency, preculture conditions, acetosyringone concentration, A. tumefaciens cell density, and microinjection/sonication parameters were evaluated. The shoot apex explants were microinjected with A. tumefaciens strain EHA105 carrying the uidA gene and co-cultivated for 3 days on mMS medium fortified with 100 mM acetosyringone. Maximum transformation efficiency of 20.25% was achieved with preculturing explants for 24 h, microinjecting three times with OD<inf>600 nm</inf> = 0.6 containing 100 mM acetosyringone, and sonicating for 20 s. Putative transformants were regenerated with 0.8 mg/L 1-naphthaleneacetic acid, 2 mg/L thidiazuron, 16% Oscillatoria acuminata extracellular extract, and 50 mg/L kanamycin. Integration was confirmed by GUS histochemical analysis, PCR, and Southern blot. Plantlets transferred from growth chamber to greenhouse showed 90% survival. This system enables rapid, efficient gene transfer in cotton. © 2020 Elsevier B.V., All rights reserved.