Polyploidy or whole genome duplication is a frequent and recurrent phenomenon in flowering plants that has played a major role in their diversification, adaptation and speciation. The adaptive success of polyploids relates to the different evolutionary fates of duplicated genes. In this study, we explored the impact of the whole genome triplication (WGT) event in the Brassiceae tribe on the genes involved in the self-incompatibility (SI) signalling pathway, a mechanism allowing recognition and rejection of self-pollen in hermaphrodite plants. By taking advantage of the knowledge acquired on this pathway as well as of several reference genomes in Brassicaceae species, we determined copy number of the different genes involved in this pathway and investigated their structural and functional evolutionary dynamics. We could infer that whereas most genes involved in the SI signalling returned to single copies after the WGT event (i.e. ARC1, JDP1, THL1, THL2, Exo70A01) in diploid Brassica species, a few were retained in duplicated (GLO1 and PLDα) or triplicated copies (MLPK). We also carefully studied the gene structure of these latter duplicated genes (including the conservation of functional domains and active sites) and tested their transcription in the stigma to identify which copies seem to be involved in the SI signalling pathway. By taking advantage of these analyses, we then explored the putative origin of a contrasted SI phenotype between two Brassica rapa varieties that have been fully sequenced and shared the same S-allele (S60).