Karyotypes of three accessions of Hordeum chilense (H1, H16, and H7), Hordeum vulgare, and Triticum aestivum were characterized by physical mapping of several repetitive sequences. A total of 14 repetitive sequences were used as probes for fluorescence in situ hybridization (FISH) with the aim of identifying inter- and intraspecies polymorphisms. The (AG)12 and 4P6 probes only produced hybridization signals in wheat, the BAC7 probe only hybridized to the centromeric region of H. vulgare, and the pSc119.2 probe hybridized to both wheat and H. chilense, but not to H. vulgare. The remaining repetitive sequences used in this study produced a hybridization signal in all the genotypes. Probes pAs1, pTa-535, pTa71, CCS1, and CRW were much conserved, showing no significant polymorphism among the genotypes studied. Probes GAA, (AAC)5, (CTA)5, HvT01, and pTa794 produced the most different hybridization pattern. We identified large polymorphisms in the three accessions of H. chilense studied, supporting the proposal of the existence of different groups inside species of H. chilense. The set of probes described in this work allowed the identification of every single chromosome in all three species, providing a complete cytogenetic karyotype of H. chilense, H. vulgare, and T. aestivum chromosomes, which could be useful in wheat and tritordeum breeding programs.