Eukaryotic chromosomes can be studied either fixed on slides by use of cytogenetic techniques or through flow-cytometry. The latter enables not only the chromosome characterization but allows for the sorting and manipulation of individual chromosomes out of a complete genome, provided that target chromosomes have flow-cytometric distinctive features (e.g. size and/or DNA base content). These requirements represent a major constraint when the chromosomes of a given species are similarly sized, a common feature in plants. In wheat, this constraint can be overcome using special aneuploid mutants, each containing single arm pairs for a given chromosome of the complement, thus keeping a balanced genome composition while showing a complement with chromosome parts which are half-sized in respect to standard autosomes. However, such genotypes are available in hexaploid wheat (Triticum aestivum) exclusively in the background of Chinese Spring, a non-elite reference variety. Notably, aneuploids are not available for the most part of plants and animals. In order to overcome this major hurdle we have developed a reliable, fast and cost-effective method for Fluorescence labeling and In situ Hybridization of chromosomes In Suspension (FISHIS). The method makes use of fluorescent oligonucleotides of simple repetitive DNA sequences (SSR) or short DNA fragments, as probes, and allows for specific chromosome flow-sorting based on the hybridization pattern determined by the FISHIS probe. We have successfully applied the FISHIS methodology for flow karyotyping and sorting highly pure, single-type chromosome fractions of commercial varieties of bread and durum wheat and other Triticeae species. Moreover, the complete chromosomal sets corresponding to the two genomes (A and B) of durum wheat have also been clearly separated by the same FISHIS approach. Given the ubiquitous occurrence in eukaryotic genomes of the type of sequences used as probes, the abundance of their variants and their overall chromosome-specific distribution, their use in FISHIS experiments provides simple and fast access to individual chromosomes of virtually any eukaryotic genome, paving the way for gaining knowledge of great potential impact on basic and applied research aspects. © Springer Science+Business Media Dordrecht 2014