This paper shows the results of effective uses of a molecular cytogenetics toolbox and molecular marker to transfer leaf rust resistance genes from Aegilops tauschii × Secale cereale (DDRR, 2n = 4x = 28) amphiploid forms to triticale cv. Bogo (AABBRR, 2n = 6x = 42). The molecular markers of resistance genes and in situ hybridization analysis of mitotic metaphase of root meristems confirmed the stable inheritance of chromosome 3D segments carrying Lr32 from the BC2F2 to the BC2F5 generation of (Ae. tauschii × S. cereale) × triticale hybrids. The chromosome pairing analysis during metaphase I of meiosis of BC2F4 and BC2F5 hybrids showed increasing regular bivalent formation of 3D chromosome pairs and decreasing number of univalents in subsequent generations. The results indicate that using amphiploid forms as a bridge between wild and cultivated forms can be a successful technology to transfer the D-genome chromatin carrying leaf rust resistance genes into triticale.