One of the many advantages of Drosophila melanogaster as a model organism is the relative ease with which gene deletions can be generated by imprecise excision of transposon insertions. Here, we describe a simple, fast, and efficient method of screening for single-gene excision events that is not biased by prior assumptions of the mutant phenotype. DNA sequence polymorphisms were used as co-dominant electrophoretic markers to identify candidate deletions in a single generation, and to delimit the breakpoints to within 0.5–1 kb, thereby rapidly identifying deficiencies that affect only the gene of interest. In addition, we used polymorphism profiling to map existing deficiencies. The method can also be applied to map the extent of deletions generated by x-rays and to identify targeted mutations generated by engineered zinc-finger nucleases in Drosophila and other polymorphic model organisms (e.g., zebrafish, mouse, Caenorhabditis elegans).