Self-incompatibility (SI) in Brassicaceae vegetables prevents self-pollination by recognizing self-pollens and rejecting them at the stigmatic surfaces. The S-haplotypes of 47 hybrid radish cultivars that are commercially available in Korea were classified and identified using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Twelve kinds of S-haplotypes were identified from the cultivars: S1, S8, S11, S17, S18, S30, and S31 haplotypes in class-I S-haplotype and S4, S5, S13, S21, and S26 haplotypes in class-II S-haplotypes. Even though the class-II S-haplotypes are supposed to exhibit weak and/or leaky SI activity, the class-II S-haplotypes showed the same allele frequency of class-I S-haplotypes in 38 fully classified commercial cultivars. The SI activity was examined using the pollen tube germination test, flower pollination test, and the seed set ratio analysis. The pollen tube test showed low correlation (R2 = 0.13) with the flower pollination test, a conventional method. The results of seed set ratio analysis varied from 0% to 159%, and thus could distinguish the weak and strong SI activity clearly and showed high correlation with the flower pollination test (R2 = 0.69). The seed set ratios of the cultivars possessing the class-I/class-I, class-I/class-II, and class-II/class-II genotypes were 0.6%, 17.4%, and 38.1%, respectively. Among the eight class-II/class-II cultivars, three cultivars showed strong SI activity. The SI activity of the S4S17, S5S8, and S4S26 genotypes varied among cultivars, but the S1S17, S5S17, and S8S26 genotypes showed constant strong, intermediate, and strong activity, respectively, among the cultivars. Results indicate that the SI activity of Brassicaceae vegetables depends not only on the S-haplotypes, but also on the genetic background of cultivars.