Fragaria vesca L., a diploid (2n = 2x = 14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6-7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l(-1) hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14-15 weeks to complete its life cycle in different seasons in the greenhouse.