Wheat expressed sequence tags (wESTs) were identified in a genomic interval predicted to span the Lr34/Yr18 slow rusting region on chromosome 7DS and that corresponded to genes located in the syntenic region of rice chromosome 6 (between 2.02 and 2.38 Mb). A subset of the wESTs was also used to identify corresponding bacterial artificial chromosome (BAC) clones from the diploid D genome of wheat (Aegilops tauschii). Conservation and deviation of micro-colinearity within blocks of genes were found in the D genome BACs relative to the orthologous sequences in rice. Extensive RFLP analysis using the wEST derived clones as probes on a panel of wheat genetic stocks with or without Lr34/Yr18 revealed monomorphic patterns as the norm in this region of the wheat genome. A similar pattern was observed with single nucleotide polymorphism analysis on a subset of the wEST derived clones and subclones from corresponding D genome BACs. One exception was a wEST derived clone that produced a consistent RFLP pattern that distinguished the Lr34/Yr18 genetic stocks and well-established cultivars known either to possess or lack Lr34/Yr18. Conversion of the RFLP to a codominant sequence tagged site (csLV34) revealed a bi-allelic locus, where a variant size of 79 bp insertion in an intron sequence was associated with lines or cultivars that lacked Lr34/Yr18. This association with Lr34/Yr18 was validated in wheat cultivars from diverse backgrounds. Genetic linkage between csLV34 and Lr34/Yr18 was estimated at 0.4 cM.