Technical Abstract: Transposable elements (TE) exist in the genomes of nearly all eukaryotes. TE mobilization through “cut-and-paste” or “copy-and-paste” mechanisms causes their insertions into other repetitive sequences, gene loci, and other DNA. An insertion of a TE produces a junction consisting of the TE-end sequence on one side and the sequence of the insertion site on the other side. Most of these junctions are unique in a genome. Because of rapid amplification of TEs and their fast turnover, the junctions are also randomly distributed along chromosomes. TE-based junctions are therefore useful for genome-wide marker development in large and complex genomes such as wheat. Several TE-based marker systems have been developed and applied to genetic diversity assays, and to genetic and physical mapping. No web-based software tool is currently available for repeat junction discovery and fully automated high-throughput repeat junction-based primer design. A software tool “RJPrimers” is reported ! here. This tool first identifies potentially unique repeat junctions using BLASTN against fully annotated repeat databases and a repeat junction finding algorithm, and then designs repeat junction primers using Primer3 and BatchPrimer3. Using in silico and wet lab PCR experiments, the software was tested based on the rice genome, as well as Sanger shotgun sequences and Roche 454 reads of the diploid wheat Aegilops tauschii. Over 90% of repeat junction primers designed by RJPrimers were unique. At least one RJM marker per 10 Kb sequence of Ae. tauschii was expected with an estimate of 0.45 million such markers in a genome of 4.5Gb, providing an almost unlimited source of molecular markers for mapping large and complex genomes. A web-based server and a command line based pipeline for RJPrimers have been implemented to meet different requirements and are available at http://wheat.pw.usda.gov/demos/RJPrimers/.