The yeast reverse two-hybrid method was developed to identify mutations disrupting protein-protein interactions. Adoption of the method has been slow, in large part, due to the high frequency of truncation and frameshift mutants typically observed with current protocols. We have developed a new strategy, based on in vitro recombinational cloning and full-length selection in E. coli, to eliminate this background and dramatically increase the efficiency of the reverse two-hybrid protocol. The method was tested by generating an allele library of MyoD1 and selecting for alleles with defective interaction with Id1. Our results confirm that most of the interaction defective alleles contain a single point mutation in the known interaction domain, the bHLH region. Moreover, analysis of the crystal structure of MyoD reveals the majority of these mutations occurred at the interaction interface. The results obtained using this novel approach for allele library generation demonstrate a significant advancement in the application of yeast reverse two-hybrid screens. Furthermore, this method is applicable to any loss-of-function mutant screen where truncated proteins are a source of high background.