Agropyron cristatum (L.) Gaertn, as Gaertn.one of important wheat wild relatives, provides gene resources being valuable for wheat genetic improvement. Introducing alien genes from A. cristatum into wheat can broaden the genetic basis of wheat. A series of wheat-A. cristatum introgression lines were produced in our previous studies, including line II-23 that was proved to be an addition and substitution lines with 19 pairs of wheat chromosomes（absence of chromosome 4B and 7A）and three pairs of A. cristatum chromosomes (2P, 4P and 7P). In this study, a spontaneous translocation line 7-20 was identified in the backcrossing progenies of II-23. By genome in situ hybridization (GISH) and non-denaturing FISH (ND-FISH) technologies, line 7-20 was shown to be a whole arm translocation line, and the translocation occurred on short arm of chromosome 7 (7A). By applying ten pairs of SSR markers that are targeted to the short arm of wheat chromosome 7A, three pairs of SSR markers presented polymorphism among the homozygous translocation line 7-20, A. cristatum and wheat. As indicated by marker analysis, the short arm of wheat chromosome 7AS was substituted by the alien chromosome segment. To confirm the size of alien chromosome segment from A. cristatum, EST-STS markers specific for A. cristatum chromosome 7P in wheat genetic background were used. We found that all of the markers located on the long arm of A. cristatum chromosome 7P were amplified in line 7-20, whereas other markers located on the short arm of A. cristatum chromosome 7P failed to amplify PCR products. As a result, the translocation line 7-20 was identified as a T7PL.7AL Robertsonian translocation line. Furthermore, we generated the segregation populations by crossing the translocation line 7-20 and recurrent wheat parent Fukuhokomugi, in order to decipher if the desirable agronomic traits are present in the alien chromosome segment. The plant height and the kernel number per spike of the translocation line T7PL.7AL were significantly reduced than those of non-translocation plants, while the fertile tillers and grain weight have no obvious changes. Taken together, this study confirmed that line 7-20 was a T7PL.7AL Robertsonian translocation line, which provided the possibility for utilizing alien genes from chromosome 7P of A. cristatum in wheat improvement.