New leaf rust resistance genes are needed in wheat (Triticum aestivum L.) to provide additional sources of resistance to the highly variable and dynamic leaf rust pathogen Puccinia triticina Eriks. Leaf rust resistance gene Lr51, located within a segment of Triticum speltoides Taush chromosome 1S translocated to the long arm of chromosome 1B of bread wheat, is resistant to the current predominant races of leaf rust in the USA. The objectives of this study were to determine the genetic length of the translocated 1S segment, develop a PCR marker for Lr51, and use this marker to generate isogenic lines for this gene. Characterization of two translocation lines (F-7-3 and F-7-12) with 10 molecular markers indicated that F-7-3 has an interstitial T. speltoides chromosome segment of 14 to 32 cM long including loci XAga7 and Xmwg710, whereas line F-7-12 has a complex series of translocations among chromosomes of homeologoos group 1. On the basis of the DNA sequence of the A, B, D, and S alleles of the XAga7 locus, we designed a cleavage amplified polymorphic sequence (CAPS) marker for the S genome allele. Primers S30-13L and AGA7-759R preferentially amplified the XAga7 1S (819 bp) and 1B alleles (783 bp). These amplification products can be separated in agarose gels after digestion with Art or BamHI restriction enzymes. This CAPS marker was validated in a collection of 32 common wheat cultivars and was used to develop three pairs of hard red spring isogenic lines from the donor parent F-7-3. These isogenic lines will be valuable for future assessment of the effect of this chromosome introgression on agronomic performance and end-use quality.