A new strategy for the rapid creation of DNA deletion libraries using a simple PCR-based method is presented. Unidirectional deletion fragments are created and may be cloned into any vector system without the constraint of using restriction enzymes. Our strategy combines methodologies from DNA sequencing, PCR, and homologous recombination (either in vivo or in vitro) to allow for the creation of a library containing fragments representing all possible deletions of a given cDNA. Using this strategy we have successfully constructed a deletion library of the cDNA encoding for the lumenal domain of yeast Ire1p, and have shown that resulting fragments range from 100 bp to the full length cDNA (1557 bp). This method is simple, inexpensive, and can easily be adapted for automated high-throughput research.