| Summary: | <i>Thinopyrum intermedium</i> possesses many desirable agronomic traits that make it a valuable genetic source for wheat improvement. The precise identification of individual chromosomes of allohexaploid Th. intermedium is a challenge due to its three sub-genomic constitutions with complex evolutionary ancestries. The non-denaturing fluorescent in situ hybridization (ND-FISH) using tandem-repeat oligos, including Oligo-B11 and Oligo-pDb12H, effectively distinguished the St, J and J<sup>S</sup> genomes, while Oligo-FISH painting, based on seven oligonucleotide pools derived from collinear regions between barley (<i>Hordeum vulgare</i> L.) and wheat (<i>Triticum aestivum</i> L.), was able to identify each linkage group of the <i>Th. intermedium</i> chromosomes. We subsequently established the first karyotype of <i>Th. intermedium</i> with individual chromosome recognition using sequential ND-FISH and Oligo-FISH painting. The chromosome constitutions of 14 wheat–<i>Th. intermedium</i> partial amphiploids and addition lines were characterized. Distinct intergenomic chromosome rearrangements were revealed among <i>Th. intermedium</i> chromosomes in these amphiploids and addition lines. The precisely defined karyotypes of these wheat–<i>Th. intermedium</i> derived lines may be helpful for further study on chromosome evolution, chromatin introgression and wheat breeding programs.
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