Genetic transformation in Prunus persica genotypes could add single horticultural traits in existing cultivars without modifying their commercial characteristics. Despite this opportunity, the final setting of transformation protocols in the species endures two major limiting factors preventing the development of new varieties: (a) explants are recalcitrant in regenerating adventitious transformed shoots and (b) have a limited regeneration capability, usually extended to just a few genotypes (i.e., cultivar dependence). This protocol illustrates a procedure that has allowed the establishment of transformation methodologies in several varieties of the species and is presented here for Red Top and Elegant Lady; this methodology could help extend technical baselines for successful transformation procedures in other, potentially more recalcitrant, and newly developed cultivars or genus members.