Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease worldwide. A recently emerged great threat to global wheat production is Pgt strain Ug99 and its derivatives, which have overcome most of the commonly used resistance genes. Rye (Secale cereale L.), closely related to wheat (Triticum aestivum L.), is a significant and valuable resource of resistance genes for wheat germplasm improvement. It is of great importance and urgency to identify new resistance gene sources of rye and transfer them into wheat. In this study, two complete sets of wheat-rye addition lines were established through wide hybridization, chromosome doubling and backcrossing. A wheat-rye 3RL telosomic addition line was identified with high resistance to stem rust strain Ug99. PCR-based markers specific for the rye chromosome were developed. Furthermore, abundant chromosomal aberrations such as minichromosomes, ring chromosomes as well as centromere reduction and expansion were identified in the progeny of wheat-rye addition lines by multicolor GISH and FISH. The line carrying a novel resistance gene to stem rust can be utilized as a bridge material for wheat disease resistance breeding. The chromosomal and centromeric variation within the wheat-rye hybrids can further contribute to genetic diversity of their offspring.