A number of leading crop species are either polyploid and/or hold a large genome as a result of the accumulation of highly repetitive sequences. These features hamper the assembly of complex genomes in spite of the fast development of new sequencing technologies (Next Generation Sequencing) and the availability of powerful bio-informatic tools. The chromosome approach, by enabling genome dissection into single chromosomes or chromosome arms via flow sorting, can contribute to reduce genome complexity. In all plants, this approach is restricted to species, or special cytogenetic stocks, as in the case of wheat, containing chromosome types that differ in size from the standard complement. We have developed an overall robust and easy method called FISHIS (Fluorescence In Situ Hybridization in Suspension) which overcomes the constrain of chromosome size and DNA content differences, allowing chromosome flow sorting on the base of fluorescent hybridization labeling of chromosomes in suspension. FISHIS relies on readily available synthetic, fluorescently labeled repetitive sequences and on alkaline DNA denaturation. We show that the method can discriminate between and hence isolate the A genome from the B genome of durum wheat (Triticum turgidum subsp. durum), a number of the chromosomes of the same species and of T. monococcum, as well as the whole complement of the diploid wheat relative Dasypyrum villosum (L.) Candargy.