The chromosome constitution on H96276-2, which is a stable line derived from a sterile wheat-Agropyron intermedium partial amphiploid Zhong 3 cross to common wheat Hui 75, was analyzed by sequential GISH and FISH. The results of GISH using genomic DNA of Agropyron intermedium as probe showed that H96276-2 possessed 20 pairs of wheat chromosomes and one pair of translocation chromosomes of wheat and Ag. intermedium. The translocated alien chromosome fragments located on the terminals of one pair of wheat chromosomes. Furthermore, reprobing of repeat sequence pSc119 was used to identify the wheat chromosomes involved in the translocation. It was determined that the alien fragments located in the terminals of the short arms of 2B chromosomes of wheat.