Stable introgression of agronomically important traits into crop plants through wide crossing often requires the generation and identification of translocation lines. However, the low efficiency of identifying lines containing translocations is a significant limitation in utilizing valuable alien chromatin-derived traits. Selection of putative wheatgrass-wheat translocation lines based on segregation ratios of progeny from gamma-irradiated seed using a standard phenotypic analysis resulted in a low 4% success rate of identifying barley yellow dwarf virus (BYDV) resistant and susceptible translocation lines. However, 58% of the susceptible progeny of this irradiated seed contained a Thinopyrum intermedium chromosome-specific repetitive sequence, which indicated that gamma-irradiation-induced translocations occurred at high rate. Restriction fragment length polymorphism (RFLP) analysis of susceptible lines containing alien chromatin, their resistant sister lines and other resistant lines showed that more than one third of the progeny of gamma-irradiated double monosomic seeds contained wheatgrass-wheat translocations. Genomic in situ hybridization (GISH) analysis of selected lines confirmed that these were wheatgrass-wheat translocation lines. This approach of initially identifying BYDV susceptible deletion lines using an alien chromosome-specific repetitive sequence followed by RFLP analysis of their resistant sister lines efficiently identified resistant translocation lines and localized the BYDV resistance to the distal end of the introgressed Th. intermedium chromosome.