Key message S29 haplotype does not require the MLPK function for self-incompatibility in Brassica rapa. Abstract Self-incompatibility (SI) in Brassicaceae is regulated by the self-recognition mechanism, which is based on the S-haplotype-specific direct interaction of the pollen-derived ligand, SP11/SCR, and the stigma-side receptor, SRK. M locus protein kinase (MLPK) is known to be one of the positive effectors of the SI response. MLPK directly interacts with SRK, and is phosphorylated by SRK in Brassica rapa. In Brassicaceae, MLPK was demonstrated to be essential for SI in B. rapa and Brassica napus, whereas it is not essential for SI in Arabidopsis thaliana (with introduced SRK and SP11/SCR from related SI species). Little is known about what determines the need for MLPK in SI of Brassicaceae. In this study, we investigated the relationship between S-haplotype diversity and MLPK function by analyzing the SI phenotypes of different S haplotypes in a mlpk/mlpk mutant background. The results have clarified that in B. rapa, all the S haplotypes except the S29 we tested need the MLPK function, but the S29 haplotype does not require MLPK for the SI. Comparative analysis of MLPK-dependent and MLPK-independent S haplotype might provide new insight into the evolution of S-haplotype diversity and the molecular mechanism of SI in Brassicaceae.