Abstract:Genome in situ hybridization and C-banding were applied to study the hybrids of two wheat-rye substitution lines 5A/5R and 6A/6R , the mechanism of homoeologous chromosome pairing was clarified and wheat-rye translocation lines was created. The chromosome behavior of pollen mother cell (PMCs) in meiosis was investigated in hybrid F1, homeologous chromosome pairing between wheat and rye occurred in 22.9% of PMC. Wheat-rye translocation lines were indentified via C-anding and Genome in situ hybridization (GISH) in hybrid F2 and later generations. In F2 generation, translocations happened in 9 of 45 plants, reached to 20%. These translocation lines were generated from homeologous chromosome pairing, or mis-division and reconstruction of univalent chromosomes.