Yellow spot (caused by Pyrenophora tritici-repentis) is a major foliar disease in wheat (Triticum aestivum) that has become more serious in recent years, possibly because of climate change. A major quantitative trait locus (QTL) located on the short arm of wheat chromosome 2B explaining 30–40% of the phenotypic variance has been identified as responsible for resistance to Australian yellow spot isolates, which reportedly produce mostly the ToxA effector. The closest marker linked to this QTL was a DArT marker not easy to use in large-scale selections, whereas the closest PCR-based marker available (2.7 cM) was too far away for reliably tagging the locus in wheat breeding. We therefore undertook studies to develop more closely linked and user-friendly markers for this major QTL. Forty-one new markers either synthesised from DArT markers or identified from the GrainGene database were assessed. From these, we developed a new PCR-based marker (Rfsts1), located 0.3 cM away from the major QTL. This is the first suitable marker for marker-assisted selection for yellow spot resistance in Australian wheat-breeding programs.