Flow sorting enabled to dissect the genome of crested wheatgrass into individual chromosomes. The flow sorted chromosome- or chromosome-arm-specific subgenomic DNA samples provide attractive resources to perform structural and functional genome analysis. Successful sorting of B chromosomes (Bs) of crested wheatgrass provides an opportunity to determine their gene content and reveal their origin, as well as discovering the mechanisms responsible for their maintenance into the population. This is a powerful approach to access the complex genome of a wheat wild relative. Chromosome genomics will support alien introgression breeding of the crop.