The addition of marker protein genes to respiratory syncytial virus (RSV) has enabled studies of the spread of RSV in different types of cell cultures and quantification of viral replication in those cultures. Genetic deletion of individual RSV genes from RSV genome has been used to determine their importance in virus infection and the differences between infection of cultured cells lines and of primary well-differentiated human bronchial epithelial (HBE) cultures. Modifications of individual viral proteins can identify the importance of a particular glycosylation, cleavage, or antigenic sites or reveal sites with these functions. However, the standard recombinant systems for the RSV genome, based on natural and inserted restriction sites, have been difficult to use, slow to accomplish, and frequently not successful. Here, we describe a yeast-based cDNA assembly system that streamlines both the construction of a cDNA clone of an RSV strain as well as any modification of an RSV cDNA, thereby enabling the rapid generation of an RSV mutant virus.