The wheat–Thinopyrum amphiploid ‘Agrotriticum # 3425’ (AT 3425), which is highly resistant to Cephalosporium stripe, was identified to carry seven pairs of Thinopyrum chromosomes, three pairs of wheat–Thinopyrum translocated chromosomes and 18 pairs of wheat chromosomes. Fluorescence genomic in situ hybridization (FGISH), C-banding, sequential C-banding and FGISH, and denaturing polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize and identify the chromosomes. The Thinopyrum chromosomes in AT 3425 were designated as T1 through T7 based on their C-banding patterns. The FGISH and C-banding patterns of mitotic chromosomes in AT 3425 and meiotic chromosomes in the hybrid between AT 3425 and wheat cultivar ‘Chinese Spring’ (CS) revealed that wheat chromosomes 1D, 2B and 3D were involved in the three wheat-Thinopyrum chromosome translocations designated as (W-T)1, (W-T)2, and (W-T)3 respectively. The analysis of high-molecular-weight glutenin subunits in single seeds of AT 3425 confirmed the involvement of wheat chromosome 1D in the translocation (W-T)1. The designations 1DSuu.1DL-1TL, 2BSuu.2BL-2TL and 3DSuu.3DL-3TL were suggested for the wheat–Thinopyrum translocated chromosomes (W-T)1, (W-T)2 and (W-T)3 in AT 3425 respectively.