Using two divergent nucleotide binding site (NBS) regions from wheat sequences of the NBS-LRR (leucine rich repeat) class, we retrieved 211 wheat and barley NBS-containing resistance gene analogue (RGA) expressed sequence tags (ESTs). These ESTs were grouped into 129 gene sequence groups that contained ESTs that were at least 70% identical at the DNA level over at least 200 bp. Probes were obtained for 89 of these RGA families and chromosome locations were determined for 72 of these probes using nullitetrasomic Chinese Spring wheat lines. RFLP analysis of 49 of these RGA probes revealed 65 mappable polymorphic bands in the doubled haploid Cranbrook x Halberd wheat population (C x H). These bands mapped to 49 loci in C x H. RGA loci were detected on all 21 chromosomes using the nullitetrasomic lines and on 18 chromosomes (linkage groups) in the C x H map. This identified a set of potential markers that could be developed further for use in mapping and ultimately cloning NBS-LRR-type disease resistance genes in wheat.