The multidrug and toxic compound extrusion (MATE) protein family is important in the export of toxins and other substrates, but detailed information on this family in the Brassicaceae has not yet been reported compared to Arabidopsis thaliana. In this study, we identified 57, 124, 81, 85, 130, and 79 MATE genes in A. thaliana, Brassica napus, Brassica oleracea, Brassica rapa, Brassica juncea, and Brassica nigra, respectively, which were unevenly distributed on chromosomes owing to both tandem and segmental duplication events. Phylogenetic analysis showed that these genes could be classified into four subgroups, shared high similarity and conservation within each group, and have evolved mainly through purifying selection. Furthermore, numerous B. napus MATE genes showed differential expression between tissues and developmental stages and between plants treated with heavy metals or hormones and untreated control plants. This differential expression was especially pronounced for the Group 2 and 3 BnaMATE genes, indicating that they may play important roles in stress tolerance and hormone induction. Our results provide a valuable foundation for the functional dissection of the different BnaMATE homologs in B. napus and its parental lines, as well as for the breeding of more stress-tolerant B. napus genotypes.