The synthetic oligonucleotide probes Oligo-pTa535 and Oligo-pAs1 were previously developed by other authors based on the repetitive sequences of clones pTa535 and pAs1 available in GenBank, and have been used as probes in fluorescence in situ hybridization (FISH) experiments allowing the identification of wheat and rye chromosomes. Their suitability as nondenaturing fluorescence in situ hybridization (ND-FISH) probes turned the hybridization procedure less time consuming, easier and more affordable. Contrastingly to conventional FISH, ND-FISH does not require DNA denaturing, preserving the chromosomal morphology and being adequate for successive hybridizations on the same chromosome spread. In this study, we hybridized, for the first time, the Oligo-pTa535 and Oligo-pAs1 probes on mitotic chromosomes of hexaploid tritordeum using ND-FISH. Tritordeum (HchHchAABB) is the synthetic amphiploid resultant from crosses between wild barley (HchHch) and cultivated durum wheat (AABB). Both Oligo-pTa535 and Oligo-pAs1 hybridized on all H. chilense-origin chromosomes of tritordeum, allowing their identification and the construction of an ideogram based on their hybridization patterns and on the physical location of the 45S ribosomal DNA (rDNA) loci detected by pTa71. We are confident that this ideogram will be further useful for the identification of H. chilense-origin chromosomes in other tritordeum lines or allopolyploids involving the Hch-genome.