Chromosomes 5A (u) , 5S and 5D can be isolated from wild progenitors, providing a chromosome-based approach to develop tools for breeding and to study the genome evolution of wheat. The three subgenomes of hexaploid bread wheat originated from Triticum urartu (A(u)A(u)), from a species similar to Aegilops speltoides (SS) (progenitor of the B genome), and from Ae. tauschii (DD). Earlier studies indicated the potential of chromosome genomics to assist gene transfer from wild relatives of wheat and discover novel genes for wheat improvement. This study evaluates the potential of flow cytometric chromosome sorting in the diploid progenitors of bread wheat. Flow karyotypes obtained by analysing DAPI-stained chromosomes were characterized and the contents of the chromosome peaks were determined. FISH analysis with repetitive DNA probes proved that chromosomes 5A(u), 5S and 5D could be sorted with purities of 78-90 %, while the remaining chromosomes could be sorted in groups of three. Twenty-five conserved orthologous set (COS) markers covering wheat homoeologous chromosome groups 1-7 were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. These assays validated the cytomolecular results as follows: peak I on flow karyotypes contained chromosome groups 1, 4 and 6, peak II represented homoeologous group 5, while peak III consisted of groups 2, 3 and 7. The isolation of individual chromosomes of wild progenitors provides an attractive opportunity to investigate the structure and evolution of the polyploid genome and to deliver tools for wheat improvement.