Two E genome--(including Ee and Eb) and two St-genome-specific RAPD markers were successfully cloned. Sequencing data indicated that the four DNA fragments were reported for the first time. Chromosome fluorescent in situ hybridization (FISH) showed that OPD-12(444) (E-specific) was a highly tandem repeat. OPF-03(1296) (Eb-specific), OPB-08(525) (St-specific), and OPN-01(817) (St-specific) were highly dispersed repetitive sequences. The FISH and Southern hybridization of genomic DNA also revealed that all of the four highly repetitive sequences were shared among relatively closer genomes of triticeae. The difference was mainly in the fragment length and the copy number. Whether a genome can amplify a specific sequence via RAPD is mainly dependent on if there are primering sites at both ends of this sequence in the genome. We also discussed the potentiality of these highly repetitive DNA fragments in detection of alien chromatin in wheat and determination of genome constitution of polyploid triticeae species.