SummaryTo assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T‐DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T‐DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T‐DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high‐quality template DNA accelerates the identification of T‐DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T‐DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T‐DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T‐DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.