The large collections of Arabidopsis thaliana sequence-indexed T-DNA insertion mutants are among the most important resources to emerge from the sequencing of the genome. Several laboratories around the world have used the Arabidopsis reference genome sequence to map T-DNA flanking sequence tags (FST) for over 325,000 T-DNA insertion lines. Over the past decade, phenotypes identified with T-DNA-induced mutants have played a critical role in advancing both basic and applied plant research. These widely used mutants are an invaluable tool for direct interrogation of gene function. However, most lines are hemizygous for the insertion, necessitating a genotyping step to identify homozygous plants for the quantification of phenotypes. This situation has limited the application of these collections for genome-wide screens. Isolating multiple homozygous insert lines for every gene in the genome would make it possible to systematically test the phenotypic consequence of gene loss under a wide variety of conditions. One major obstacle to achieving this goal is that 12% of genes have no insertion and 8% are only represented by a single allele. Generation of additional mutations to achieve full genome coverage has been slow and expensive since each insertion is sequenced one at a time. Recent advances in high-throughput sequencing technology open up a potentially faster and cost-effective means to create new, very large insertion mutant populations for plants or animals. With the combination of new tools for genome-wide studies and emerging phenotyping platforms, these sequence-indexed mutant collections are poised to have a larger impact on our understanding of gene function.