Wheat (Triticum aestivum) and jointed goatgrass (Aegilops cylindrica) can cross with each other, and their self-fertile backcross progenies frequently have extra chromosomes and chromosome segments, presumably retained from wheat, raising the possibility that a herbicide resistance gene might transfer from wheat to jointed goatgrass. Genomic in situ hybridization (GISH) was used to clarify the origin of these extra chromosomes. By using T. durum DNA (AABB genome) as a probe and jointed goatgrass DNA (CCDD genome) as blocking DNA, one, two, and three A- or B-genome chromosomes were identified in three BC2S2 individuals where 2n = 29, 30, and 31 chromosomes, respectively. A translocation between wheat and jointed goatgrass chromosomes was also detected in an individual with 30 chromosomes. In pollen mother cells with meiotic configuration of 14 II + 2 I, the two univalents were identified as being retained from the A or B genome of wheat. By using Ae. markgrafii DNA (CC genome) as a probe and wheat DNA (AABBDD genome) as blocking DNA. 14 C-genome chromosomes were visualized in all BC2S2 individuals. The GISH procedure provides a powerful tool to detect the A or B-genome chromatin in a jointed goatgrass background, making it possible to assess the risk of transfer of herbicide resistance genes located on the A or B genome of wheat to jointed goatgrass.