DNA polymorphisms are valuable for several applications including genotyping, molecular mapping and marker-assisted selection. The 55 K Affymetrix Wheat GeneChip was used to survey expression level polymorphisms (ELPs) and single-feature polymorphisms (SFPs) between two near-isogenic wheat genotypes (BC(7):F(4)) that differ for the Yr5 stripe rust resistance locus, with the objective of developing genetic markers linked to Yr5. Ninety-one probe sets showing ELPs and 118 SFP-containing probe sets were identified between isolines, of which just nine ELP probe sets also contained SFPs. The proportion of the transcriptome estimated to be variable between isolines from this analysis was 0.30% for the ELPs and 0.39% for the SFPs, which was highly similar to the theoretical genome difference between isolines of ~0.39%. Using wheat-rice synteny, both ELPs and SFPs mainly clustered on long arms of rice chromosomes four and seven, which are syntenous to wheat chromosomes 2L (Yr5 locus) and 2S, respectively. The strong physical correlation between the two types of polymorphism indicated that the ELPs may be regulated by cis-acting DNA polymorphisms. Twenty SFPs homologous to rice 4L were used to develop additional genetic markers for Yr5. Physical mapping of the probe sets containing SFPs to wheat chromosomes identified nine on the target chromosome 2BL, thus wheat-rice synteny greatly enhanced the selection of SFPs that were located on the desired wheat chromosome. Of these nine, four were converted into polymorphic cleaved amplified polymorphic sequence (CAPS) markers between Yr5 and yr5 isolines, and one was mapped within 5.3 cM of the Yr5 locus. This study represents the first array-based polymorphism survey in near-isogenic genotypes, and the results are applied to an agriculturally important trait.