The chromosomal composition of the spring triticale line 131/7 carrying a rye-wheat translocation was studied using GISH, C-banding, and SSR analysis. The complex analysis revealed the presence of a pair of 2D chromosomes and T2RS.2RL-2BL translocation in the genome of the hexaploid triticale line 131/7. The break point in the translocated chromosome is between the markers Xwmc592 and Xwmc441, loci 63.9 cM and 76.8 cM on the linkage map (Wheat, Consensus SSR, 2004 NA-SSR-2004-2B) and in the region C2BL2-0.36 on the physical map (Wheat, Physical, SSR). The analysis of the line enables its use in breeding programs and in genetic studies.