Self-incompatibility (SI) systems are widespread mechanisms that prevent self-fertilization in angiosperms. They are generally encoded by one genome region containing several multiallelic genes, usually called the S-locus. They involve a recognition step between the pollen and the pistil component and pollen is rejected when it shares alleles with the pistil. The direct consequence is that rare alleles are favored, such that the S-alleles are subject to negative frequency-dependent selection. Several theoretical articles have predicted the specific patterns of polymorphism, compared to neutral loci, expected for such genes under balancing selection. For instance, many more alleles should be maintained and populations should be less differentiated than for neutral loci. However, empirical tests of these predictions in natural populations have remained scarce. Here, we compare the genetic structure at the S-locus and microsatellite markers for five natural populations of the rare species Brassica insularis. As in other Brassica species, B. insularis has a sporophytic SI system for which molecular markers are available. Our results match well the theoretical predictions and constitute the first general comparison of S-allele and neutral polymorphism.H OMOMORPHIC self-incompatibility (SI) systems are widespread physiological mechanisms preventing self-fertilization in Angiosperms by controlling pollen germination or pollen tube growth (De Nettancourt 2001). Pollen and pistil are incompatible when they both express identical alleles. The recognition involves specificity molecules usually encoded by one genome region containing several multiallelic genes (De Nettancourt 2001). In gametophytic (GSI) systems, the pollen phenotype is encoded by its own haploid genome, whereas in sporophytic (SSI) systems, the pollen phenotype is determined by the sporophyte (diploid pollen parent) and can involve dominance interactions among alleles. For instance, two classes of alleles are known in Brassica oleracea (Nasrallah 1991). Class I alleles are dominant over the class II alleles in the pollen, while alleles within class I and class II are mutually codominant. In the pistil, all alleles are codominant. This scheme is also found in B. campestris, with the exception that alleles occur in three dominance levels in the pollen and a few alleles are recessive in the stigma (Hatakeyama et al. 1998). At the molecular level, the SSI system of the Brassicaceae is among the best known (for a recent review see Hiscock and McInnis 2003). Both pistil and pollen genes have been identified (Schopfer et al. 1999;Takayama et al. 2000). In this system, recognition proceeds through receptor-ligand interaction between S-locus cysteinerich protein (SCR), a small hypervariable ligand peptide, and S-locus receptor kinase (SRK), a transmembrane receptor with an intracellular kinase domain (Kachroo et al. 2001).SI evolutionary properties have also long aroused the population geneticist's interest because selection pressures are known a priori. C...