Nineteen strains of Drosophila virilis from diverse geographic locations were examined by genetic and molecular analyses, revealing (a) 12 strains with a single copy of the urate oxidase (UO) gene per haploid genome and (b) 7 strains with a tandem duplication of the UO locus. The D. virilis strains with the UO duplication appear to have identical restriction maps of this region, implying either a single origin for the duplication or several similar events occurring at a hot spot. On the basis of the location of the duplication breakpoints and the restriction sites flanking these breakpoints, this duplication probably arose through nonhomologous recombination involving either a breakage and rejoining event or replication slippage. because documented cases of intraspecific gene duplication polymorphism are rare, the D. virilis UO duplication will be useful in identifying the molecular event giving rise to a gene duplication.