A 2RL.2BS wheat-rye translocation, present in the wheat germplasm line Hamlet, carries a gene for resistance to Hessian fly biotype L, one of the most virulent biotypes presently encountered in wheat production environments. Unlike several other wheat-rye chromosome translocations common in wheat breeding programs, 2RL lacks genes encoding storage proteins or other easily selected markers. Oligonucleotide primers synthesized from published sequences derived from the R173 family of moderately repetitive rye DNA were used in the DNA polymerase chain reaction (PCR) to identify specific markers for 2RL. The same primers, when used with DNA extracted from additional wheat-rye translocation lines of importance to the wheat breeding community, gave distinctive PCR products for each genotype. The single primer pair, PAWS5 and PAWS6, may, therefore, have wide applicability for the identification of wheat-rye chromosomal translocations presently encountered in wheat breeding populations.