Rust resistance genes Lr37, Sr38, and Yr17 are located within a segment of Triticum ventricosum (Tausch) Cess. chromosome 2NS translocated to the short arm of bread wheat chromosome 2AS. Characterization of this chromosome segment by 13 restriction fragment length polymorphism (RFLP) markers indicated that the 2NS translocation replaced approximately half of the short arm of chromosome 2A (distal 25-38 centimorgans, cM). The objective of this study was to develop polymerase chain reaction (PCR) assays based on RFLP marker cMWG682 to facilitate the transfer of this cluster of rust resistance genes into commercial wheat (Triticum aestivum L.) cultivats. DNA sequence was obtained from the A-, B-, D-, and N-alleles of cMWG682 and was used to design N-allele specific primers. The 2NS fragment amplified by PCR primers cosegregated with the presence of the RFLP-2NS band in all backcross populations. A cleaved amplified polymorphic sequence (CAPS) was used to develop a marker for the 2A-allele. This marker can be used to differentiate homozygous and heterozygous plants carrying the 2NS translocation in the final cycle of backcross introgression or in screenings for homozygous plants in segregating populations. Finally, a third PCR assay was developed by means of TaqMan technology as a high-throughput alternative for selection of the 2NS/2AS translocation in large segregating populations in breeding programs that have access to real time PCR equipment. These molecular markers were used to develop four hard red spring isogenic lines homozygous for the 2NS chromosome segment. One of the isogenic lines, derived from 'Anza,' did not show the expected resistance in spite of the presence of all the RFLP markers for the 2NS chromosome segment. Analysis of F1 hybrids suggested that a suppressor of the Lr37 gene is present in Anza. These isogenic lines will provide a valuable tool to test the effects of the large 2NS translocation on quality and agronomic performance.