Abstract This article aims to study the homoeologous chromosome pairing and translocation induced by 5A/5R x 6A/6R wheat-rye substitution lines. To clarify the mechanism of homoeologous chromosome pairing and create wheat-rye translocation lines, two wheat-rye substitution lines, 5A/5R and 6A/6R were crossed. The chromosome behavior of pollen mother cells (PMCs) in meiosis was investigated in hybrid F 1. Homoeologous chromosome pairing between wheat and rye occurred in 22.91% of PMC. Wheat-rye translocation lines were identified via C-banding and in situ hybridization (GISH) in hybrid F 2 and later generations. In F 2 generation, translocations happened in 9 of 45 plants, reached to 20%. These translocation lines were generated from homoeologous chromosome pairing, or mis-division and reconstruction of univalent chromosomes.