<jats:title>Summary</jats:title><jats:p>Goat grasses (<jats:italic>Aegilops</jats:italic> spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative <jats:styled-content style="fixed-case">DNA</jats:styled-content> content among chromosomes within a karyotype. This work describes the distribution of <jats:styled-content style="fixed-case">GAA</jats:styled-content> and <jats:styled-content style="fixed-case">ACG</jats:styled-content> microsatellite repeats on chromosomes of the U, M, S and C genomes of <jats:italic>Aegilops</jats:italic>, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence <jats:italic>in situ</jats:italic> hybridization (<jats:styled-content style="fixed-case">FISHIS</jats:styled-content>). Bivariate flow cytometric analysis of chromosome <jats:styled-content style="fixed-case">DAPI</jats:styled-content> fluorescence and fluorescence of <jats:styled-content style="fixed-case">FITC</jats:styled-content>‐labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. <jats:styled-content style="fixed-case">DNA</jats:styled-content> of purified chromosomes was used as a template for polymerase chain reation (<jats:styled-content style="fixed-case">PCR</jats:styled-content>) using Conserved Orthologous Set (<jats:styled-content style="fixed-case">COS</jats:styled-content>) markers with known positions on wheat A, B and D genomes. Wheat–<jats:italic>Aegilops</jats:italic> macrosyntenic comparisons using <jats:styled-content style="fixed-case">COS</jats:styled-content> markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the <jats:italic>Aegilops</jats:italic> genomes, and the <jats:styled-content style="fixed-case">COS</jats:styled-content> markers assigned to <jats:italic>Aegilops</jats:italic> chromosomes will facilitate alien gene introgression into wheat.</jats:p>