A major task in the second phase of the genome sequencing projects is the identification of coding sequence within the three billion base pairs of each the mouse and human genomes. At present, in addition to computer-aided programs, several high-throughput mutagenesis programs are being undertaken worldwide in order to achieve this goal (Ref. 1). Gene-trap mutagenesis screens take advantage of random insertions into transcription units to drive a selection–reporter cassette and simultaneously to mutate the tagged genes. One of the advantages of gene-trap mutagenesis is that no a priori knowledge is needed about the structure of the tagged gene. However, because most gene-trap vectors contain splice-acceptor or splice-donor sites to capture translated gene sequence, the precise location of vector integration within introns is not known. Therefore, it is often difficult to generate external probes for Southern blots or external primers for PCR analysis in order to distinguish between homozygous and heterozygous mutants. To overcome this serious limitation in high-throughput mutagenesis screens, we developed a real-time PCR strategy that allows us to discriminate between mutants with either one or two copies of the gene-trap vector inside their genomes. Real-time quantitative PCR is based on the quantification of a fluorescent dye [5′-6-carboxyfluorescein (5′-FAM)] that is quenched by 3′-6-carboxy-tetramethylrhodamine (3′ TAMRA) when attached to a probe located between two PCR primers but is activated by the 5′ exonuclease activity of the Taq DNA polymerase (Ref. 2,3 ). Here, we describe a rapid method based on the quantification of a gene-trap vector relative to a standard locus within the mouse genome. This method allows the rapid genotyping of any gene-trap animal without prior knowledge of the mutated genes. Here, as an example, we describe the genotyping of a PT1βgeo insertion (Ref. 4) into the mouse Neurochondrin gene (Ref. 5) by comparing a multiplex PCR assay and a novel real-time-PCR-based method.