Switchgrass is one of the most important biomass/bioenergy crops. For its improvement as a feedstock through biotechnological approach, we have developed a high throughput Agrobacterium-mediated transformation system for cv. Alamo and two new elite cultivars, Performer and Colony. Highly regenerable and transformation-competent embryogenic calli were identified and used for genetic transformation. GFP reporter gene was employed to identify transformation events at early stages and to guide modifications at various stages for improvement of transformation efficiency. The modifications included infection under vacuum, co-cultivation at desiccation conditions, resting between co-cultivation and selection, and supplement of L-proline in the callus culture and selection media. Transformation efficiency over 90% was routinely achieved for Performer, and around 50% for Alamo and Colony. The new system substantially improved switchgrass transformation efficiency and will significantly contribute to the genetic improvement of this important biofuel feedstock via biotechnological approach. (C) 2010 Elsevier Ltd. All rights reserved.