Heterostyly is a plant breeding system occurring in approximately 28 plant families and it has often been used as a model system in plant genetics and evolution. Although heterostyly has been studied for over a century beginning with Charles Darwin, the genes determining floral architecture and incompatibility are still unknown. To identify the genes residing at the S-locus of distylous Turnera subulata, we used a positional cloning strategy and assembled three BAC contigs across the S-locus region. In total, 31 overlapping BAC clones were assembled into contigs 1, 2 and SL. We developed and mapped numerous co-dominant markers from the ends of BAC clones across the S-locus region and assayed X-ray deletion mutants to delimit the region of the contig containing the S-locus. Deletion mapping revealed that a single BAC clone (L22s) within contig-SL contains the s haplotype, while two additional BAC clones (I1 and K15) may contain parts of the dominant S haplotype. Furthermore, we exploited the contigs assembled and investigated the rates of recombination at the S-locus as well as in two regions on either side of the S-locus. We found that recombination rates (estimated in kb/cM) are 2-5 times lower at the S-locus relative to flanking regions, although they are not statistically significant. The present study represents a landmark in the molecular characterization of the S-locus of a heterostylous species. We are now on the verge of identifying the genes that have remained elusive since Darwin's comprehensive study of heterostylous systems more than 125 years ago.