We have generated a new set of transgenic fly stocks that carry >80 kb BAC genomic clones tiling the majority of the autosomal portion of the Drosophila melanogaster genome. As a new fly stock collection carrying molecularly-defined genomic duplications, it is a very useful tool for rescue and complementation testing, genetic fine mapping, and can form the basis for further genomic engineering. Specifically, a set of about 1,200 overlapping BACs that tile the entirety of chromosomes 2 and 3 was selected and inserted using site-specific integration. This collection covers >99% of the genes in the autosomal genome. Together with the existing genomic duplication collections for the X and 4th chromosomes, the vast majority of the Drosophila genome is now covered. Due to the precise nature of this new collection where the end points of each BAC and the genomic insertion site are precisely known, additional applications are enabled, such as clonal analysis. Furthermore, this collection can serve as the starting point for other manipulations such as gene tagging, generating point mutations, and structure-function analyses. Transgenic stocks are now available through a web-based searchable database (http://genetivision.com/duplication.html).