Barley is a model plant in genomic studies of Triticeae species. However, barley's large genome size and high repetitive sequence content complicate the whole-genome sequencing. The majority of the barley genome is composed of transposable elements (TEs). In this study, TE repeat junctions (RJs) were used to develop a large-scale molecular marker platform, as a prerequisite to genome assembly. A total of 10.22 Gb of barley nonassembled 454 sequencing data were screened with RJPrimers pipeline. In total, 9,881,561 TE junctions were identified. From detected RJs, 400,538 polymerase chain reaction (PCR)-based RJ markers (RJMs) were designed across the genome, with an average of 39 markers/Mb. The utility of designed markers was tested using a random subset of RJMs. Over 94% of the markers successfully amplified amplicons, among which ~90% were genome specific. In addition to marker design, identified RJs were utilized to detect 1190,885 TEs across the genome. In gene-poor regions of the genome Gypsy elements comprised the majority of TEs (~65%), while in gene-rich regions Gypsy, Copia, and Mariner were the main transposons, each representing an average ~23% of total TEs. The numerous RJ primer pairs developed in this study will be a valuable resource for barley genomic studies including genomic selection, fine mapping, and genome assembly. In addition, the results of this study show that characterizing RJs provides insight into TE composition of species without a sequenced genome but for which short-read sequence data is available.