Molecular tagging and saturation mapping of agronomically important genes is essential to marker-assisted selection and mapped-based gene cloning. The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using positional cloning technology. Here, we compared the feasibility of bin-mapped ESTs, AFLPs in combination with bulked segregant analysis (AFLP-BSA), and the scanning of previously published maps for RFLPs and SSRs for targeting Tsn1 in two segregating populations. EST markers located within the wheat deletion bin 5BL-0.75-0.76 were first mapped in a low-resolution common wheat population derived from CS x CS-DIC 5B as either RFLPs or SSCPs. ESTs flanking Tsn1 were then mapped in a high-resolution durum wheat population derived from LDN x LDN-DIC 5B. The Tsn1 gene was delineated to a 0.75 cM interval by the flanking EST markers. Our results indicated that, for tagging Tsn1, the use of bin-mapped EST markers was at least as efficient as using AFLP-BSA and more effective than selection of SSRs and RFLPs based on comparisons with previously published maps.