White clover (Trifolium repens L.) is a forage legume of considerable economic importance in temperate agricultural systems with a strong self-incompatibility system. The molecular basis of self-incompatibility in T repens is unknown, but it is under the control of a single locus which is expressed gametophytically. To locate the self-incompatibility locus (S locus) in T. repens, we carried out cross-pollination experiments in an F1 mapping population and constructed a genetic linkage map using amplified fragment length polymorphism and simple sequence repeat markers. As the first step in a map-based cloning strategy, we locate for the first time the S locus in T. repens on a genetic linkage map, on the homoeologous linkage group pair 1(E), which is broadly syntenic to Medicago truncatula L. chromosome 1. On the basis of this syntenic relationship, the possibility that the S locus may or may not possess an S-RNase gene is discussed.