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SUMMARY Aegilopsspecies represent the most important gene pool for breeding bread wheat (Triticum aestivum). Thus, understanding the genome evolution, including chromosomal structural rearrangements and syntenic relationships amongAegilopsspecies or betweenAegilopsand wheat, is important for both basic genome research and practical breeding applications. In the present study, we attempted to develop subgenome D‐specific fluorescencein situhybridization (FISH) probes by selecting D‐specific oligonucleotides based on the reference genome of Chinese Spring. The oligo‐based chromosome painting probes consisted of approximately 26 000 oligos per chromosome and their specificity was confirmed in both diploid and polyploid species containing the D subgenome. Two previously reported translocations involving two D chromosomes have been confirmed in wheat varieties and their derived lines. We demonstrate that the oligo painting probes can be used not only to identify the translocations involving D subgenome chromosomes, but also to determine the precise positions of chromosomal breakpoints. Chromosome painting of 56 accessions ofAe. tauschiifrom different origins led us to identify two novel translocations: a reciprocal 3D‐7D translocation in two accessions and a complex 4D‐5D‐7D translocation in one accession. Painting probes were also used to analyze chromosomes from more diverseAegilopsspecies. These probes produced FISH signals in four different genomes. Chromosome rearrangements were identified inAegilops umbellulata,Aegilops markgrafii, andAegilops uniaristata, thus providing syntenic information that will be valuable for the application of these wild species in wheat breeding.