In Brassica, self-incompatibility (SI) can be overcome by CO2 application, an effective method for obtaining numerous inbred lines for F 1 commercial seed. We previously reported two different S-alleles of Brassica campestris, S733 and S734, with extremely different degrees of susceptibility to this gas. In the current study, we raised a cross-population between those two genetic lines, and analyzed their reaction level of self-incompatibility to CO2 (RLSICO2). Here, all 40 of our progeny from the F1 cross-population were susceptible, maintaining high values of RLSICO2. This suggests that the susceptible line, S734, is dominant to the insusceptible line, S 733. We also generated an F2 selfing-population of each crossed progeny, S 733 ♀ S734 ♂ and S 733 ♂ S734 ♀, to assess the RLSICO2 of each individual. PCR-RFLP analysis was performed to determine the S-genotype of the F2 population. The S734 allele segregated in a theoretical ratio of the dominant trait, and the RLSICO2 was consistent with the dominance relationship. Therefore, we have now demonstrated that high RLSICO2 in B. campestris is controlled by a dominant gene.