Large collections of sequence-indexed T-DNA insertion mutants are invaluable resources for plant functional genomics. Flanking sequence tag (FST) data from these collections indicated that T-DNA insertions are not randomly distributed in the Arabidopsis thaliana genome and that there are still a fairly high number of annotated genes without T-DNA insertions. We have analyzed FST data from the FLAGdb, GABI-Kat, and SIGnAL mutant populations. The lack of detectable transcriptional activity and the absence of suitable restriction sites were among the reasons genes are not covered by insertions. Additionally, a refined analysis of FSTs to genes with annotated noncoding regions showed that transcription initiation and polyadenylation site regions of genes are favored targets for T-DNA integration. These findings have implications for the use of T-DNA in saturation mutagenesis and for our chances to find a useful knockout allele for every gene.