Genetic transformation in strawberry (Fragaria spp.) can be achieved by using the Agrobacterium-mediated procedure on leaves from in vitro proliferated shoots. Regardless of the sufficient regeneration levels achieved from leaf explants of some commercial strawberry genotypes, the regeneration of transformed strawberry plants remains difficult and seems to be strongly genotype dependent. In fact, the main factors that play an important role in the success of strawberry genetic transformation are the availability of an efficient regeneration protocol and of an appropriate selection procedure of the putative transgenic shoots. The strawberry genetic transformation protocol herein described relates to three genotypes resulted from our experience with the highest regeneration and transformation efficiency. The study includes two octoploid Fragaria × ananassa cultivars, Sveva and Calypso, and a diploid F. vesca cultivar (Alpina W.O.). All the different steps related to the leaf tissue Agrobacterium infection, coculture, and selection of regenerating adventitious shoots, as well as the following identification of selected lines able to proliferate and root on the selective agent (kanamycin), will be described.