At present, only few methods for the effective assembly of multigene constructs have been described. Here we present an improved version of the MultiRound Gateway technology, which facilitates plant multigene transformation. The system consists of two attL-flanked entry vectors, which contain an attR cassette, and a transformation-competent artificial chromosome based destination vector. By alternate use of the two entry vectors, multiple transgenes can be delivered sequentially into the Gateway-compatible destination vector. Multigene constructs that carried up to seven transgenes corresponding to more than 26 kb were assembled by seven rounds of LR recombination. The constructs were successfully transformed into tobacco plants and were stably inherited for at least two generations. Thus, our system represents a powerful, highly efficient tool for multigene plant transformation and may facilitate genetic engineering of agronomic traits or the assembly of genetic pathways for the production of biofuels, industrial or pharmaceutical compounds in plants.