Genetic diversity of 180 Brassica nigra (L.) Kochgenotypes from 60 different accessions was evaluated using 15 simple sequence repeat markers with known locations on the Brassica A, B, and C genomes. Two lines each from Brassica juncea (L.) Czern and Brassica carinata Braunwere also included as comparator species. A total of 218 high quality alleles were used to generate a genetic distance matrix, and clustering and multidimensional scaling analyses were used to investigate genetic relationships among the accessions. Accessions from the same country of origin tended to cluster together. Surprisingly, 13 accessions declared to be B. nigra had A- and B-genome alleles and morphology consistent with them being B. juncea, which was supported by their positioning near B. juncea in the cluster analysis. Two B. nigra accessions possessed alleles associated more closely with the A genome than the B genome, and these may be Brassica rapa L. accessions. One B. nigra accession had B- and C-genome alleles and morphology consistent with it being B. carinata. The remaining 44 accessions (73%) appeared to be truly B. nigra and formed morphologically and genetically distinct groups associated with country or region of origin, notably Ethiopia, Israel, India, and Europe. Most B. nigra accessions were highly heterozygous, consistent with their obligate outcrossing habit. This study demonstrated the value of using molecular markers with known genome locations (in this case, in the Brassica A, B, and C genomes) to confirm species identity in families such as Brassicaceae where species identification based solely on morphological characters is difficult.