SUMMARYA chromosome‐specific painting technique has been developed which combines the most recent approaches of the companion disciplines of molecular cytogenetics and genome research. We developed seven oligonucleotide (oligo) pools derivd from single‐copy sequences on chromosomes 1 to 7 of barley (Hordeum vulgare L.) and corresponding collinear regions of wheat (Triticum aestivum L.). The seven groups of pooled oligos comprised between 10 986 and 12 496 45‐bp monomers, and these then produced stable fluorescence in situ hybridization (FISH) signals on chromosomes of each linkage group of wheat and barley. The pooled oligo probes were applied to high‐throughput karyotyping of the chromosomes of other Triticeae species in the genera Secale, Aegilops, Thinopyrum, and Dasypyrum, and the study also extended to some wheat‐alien amphiploids and derived lines. We demonstrated that a complete set of whole‐chromosome oligo painting probes facilitated the study of inter‐species chromosome homologous relationships and visualized non‐homologous chromosomal rearrangements in Triticeae species and some wheat‐alien species derivatives. When combined with other non‐denaturing FISH procedures using tandem‐repeat oligos, the newly developed oligo painting techniques provide an efficient tool for the study of chromosome structure, organization, and evolution among any wild Triticeae species with non‐sequenced genomes.