Chromosomal localization of repeat variants in grasses is consistent with repeat abundance measured by our bioinformatic pipeline. Chromosomal localization of the different grass repeat variants (maize variant A, switchgrass variants B1 and B2, witchgrass variant C, and foxtail millet variant D) was determined by FISH on metaphase chromosomes of maize (Zea mays), switchgrass (Panicum virgatum), witchgrass (Panicum capillare), and foxtail millet (Setaria italica). Switchgrass variants B1 and B2 differ by a 9-bp deletion, whereas both variants differ from maize, witchgrass and foxtail millet by a 20-bp insertion. Maize and foxtail millet chromosomes hybridized only to variants A and D, respectively. Only one switchgrass chromosome hybridized to variant A (arrow), but variants B1, B2 and C labeled most chromosomes (arrowheads indicate chromosomes that showed weaker hybridization to variant C). Witchgrass chromosomes were most consistently labeled by variant C, but showed chromosome-specific hybridization to variants B1 and B2, consistent with their lower abundance in the genome. In all cases the FISH probes hybridized to the primary constriction, which is indicative of centromere localization. The percentages below the panels represent computational predictions of repeat variant ratios in each species.