The number of S haplotypes, which are estimated to be 50 in Brassica oleracea and more than 100 in Brassica rapa, can be utilized as a criterion to assess the genetic diversity of each accession within genetic resource collections, and can be lost most readily from genetic resource accessions. The finding that the recognition specificities of pollen and stigma are determined by SP11/SCR and SRK, respectively, suggests the difficulties for generation of a new S haplotype. The self-incompatibility system is utilized for the breeding of F1 hybrid cultivars of the vegetables in B. oleracea, B. rapa, and R. sativus. In F1 hybrid breeding using the self-incompatibility system, stability or strength of self-incompatibility, which sometimes depends on the S haplotypes, is required. Therefore, it is important to maintain a large number of S haplotypes as genetic resources. We are now developing a new series of S tester lines of B. oleracea, B. rapa, and R. sativus using molecular techniques for S-haplotype identification. These methods include PCR-RFLP of SLG and SRK and dot blot analysis of SP11/SCR.