Miniature inverted-repeat transposable elements (MITES) represent a large superfamily of transposons that are moderately to highly repetitive and frequently found near or within plant genes. To elucidate the organization of MITES in the barley genome, MITES were integrated into the genetic map of barley. In this report, we describe the use of MITES in amplified fragment length polymorphism (AFLP) mapping, and demonstrate their superiority over conventional AFLP mapping. Barley MITES include members of the Stowaway, Barfly, and Pangrangja families. By amplifying the flanking sequences of these MITES, a total of 214 loci were mapped from a population of 93 doubled-haploid segregating individuals between Hordeum vulgare ssp. vulgare and H. vulgare ssp. spontaneum. The 214 MITE-AFLP and 40 anchor simple sequence repeat (SSR) loci were distributed on 7 linkage groups, covering a total map distance of 1 165 cM. The average marker density on each chromosome ranged between 3.4 and 9.6 cM per locus. Only 1 MITE-based locus was frequently found to be associated with MITE loci from the same family, resulting in clusters in chromosomal subregions. In barley, it will be possible to cover the entire genome with a limited set of MITE-based primers and to build highly dense maps of specific regions.