Watermelon is an annual fruit crop that faces various biotic and abiotic stresses. Genetic engineering is an efficient tool for improving plant traits that combat environmental stresses. In this study, we have developed an improved Agrobacterium-mediated genetic transformation system for watermelon and the same was evaluated in four cultivars (Arka Manik, Arka Muthu, IIHR-14, and Sugar Baby).Agrobacterium tumefaciens strain EHA 105 bearing binary vector pCAMBIA 1301-bar was used to standardize the transformation protocol. BASTA® 6 mg−1was used to eliminate nontransformed tissues and select transformed shoots. To improve the transformation efficiency, polyamine incorporation in the medium, sonication of explant, and vacuum infiltration were tested. The highest transformation efficiency of 17.3% was achieved with spermidine (15 mg l−1) incorporation in the culture medium, sonicating the explant for 30 s followed by 2 min vacuum infiltration during Agrobacterium-mediated genetic transformation, in the cultivar Arka Manik. The shoot induction and elongation medium containing BA (1 mg l−1), leucine (5 mg l−1), spermidine (10 mg l−1) along with BASTA® were successfully used for regeneration and elongation of shoots, from cotyledonary node. The 2-month-old regenerated putative transformants were subjected for molecular analysis. Histochemical GUS (β-glucuronidase) assay, PCR and southern analysis confirmed the presence of a transgene in the obtained transgenic watermelon lines. This transformation protocol was tested in four cultivars to prove its efficiency, which further confirmed that this genotype-independent direct regeneration–based Agrobacterium transformation protocol could be further explored for improving genetic transformation of watermelon. © 2019 SAAB