Polymorphic DNA bands were identified between a near iso-isogenic line of wheat carrying both stem (Sr39) and leaf (Lr35) rust resistance genes and the recurrent line Thatcher (Tc) which lacks these genes. Both resistance genes are located on a translocated chromosomal segment derived from Aegilops speltoides and thus are genetically linked. The primers used to generate polymorphic bands were 3'-anchored inter-simple sequence repeat primers which identified genomic microsatellites with a repeated motif of 3 nucleotides in length. The primers were used singly to amplify genomic segments which were flanked by inversely orientated, closely spaced, identical microsatellite sequences. One of the polymorphic bands, a 900 base pair band, was completely linked to the Sr39 and Lr35 rust resistance genes in the segregating population used in this study. After cloning and sequencing this polymorphic band, the inter-simple sequence repeat marker was converted to a sequence characterized amplified region marker by designing primer sets which amplify a single, easily resolved band from DNA of plants with Sr39/Lr35 genes. This marker is present in six wheat lines carrying the Sr39 and Lr35 genes on the translocated chromosome segment from Ae. speltoides. The marker has facilitated efforts to breed Canada Prairie Spring and Canada Western Extra Strong lines with these rust resistance genes