The large scale sequencing of insertion element flanking sequences has revolutionized reverse genetics in plant research: Insertion mutants can now simply be identified in silico by BLAST searching the resulting flanking sequence databases. The development of next-generation sequencing technologies has further facilitated the creation of flanking sequence collections derived from entire mutant populations. Here we describe a highly efficient and widely applicable method that we developed to amplify, sequence, and identify dTph1 transposon flanking sequences from a library of 1000 Petunia W138 individuals simultaneously.