A major QTL for resistance to Fusarium head blight (FHB) in wheat, Qfhs.ndsu-3BS, has been identified and verified by several research groups. The objective of this study was to increase the marker density in this QTL region using STS (sequence-tagged site) markers developed from wheat expressed sequence tags (ESTs) near Qfhs.ndsu-3BS. Because wheat chromosome 3BS and rice chromosome 1S are syntenous, the sequences of P1-derived artificial chromosome (PAC) and (or) bacterial artificial chromosome (BAC) clones covering the sub-distal portion of rice chromosome 1S were used as queries for a BLASTn search to identify wheat ESTs most likely near Qfhs.ndsu-3BS. Sixty-eight out of 79 STS primer pairs designed from wheat ESTs amplified PCR products from the genomic DNA of Triticum aestivum 'Chinese Spring'. Twenty-eight STS markers were localized on chromosome 3BS by aneuploid analysis. Six out of the nine STS markers that could be mapped in the T. aestivum 'Sumai 3'/T. aestivum 'Stoa' population had higher R2 and LOD values for this QTL than the most significant marker reported previously. Therefore, leveraging genome sequence information available in rice for wheat genetics is an effective strategy to develop DNA markers for Qfhs.ndsu-3BS, and this strategy may have broad applications for targeted mapping of other traits in cereal crops.