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The genus <i>Brassica</i> includes some of the most important vegetable and oil crops worldwide. Many <i>Brassica</i> seeds (which can show diagnostic characters useful for species identification) were recovered from two archaeological sites in northern Italy, dated from between the Middle Ages and the Renaissance. We tested the combined use of archaeobotanical keys, ancient DNA barcoding, and references to ancient herbarium specimens to address the issue of diagnostic uncertainty. An unequivocal conventional diagnosis was possible for much of the material recovered, with the samples dominated by five <i>Brassica</i> species and <i>Sinapis</i>. The analysis using ancient DNA was restricted to the seeds with a <i>Brassica</i>-type structure and deployed a variant of multiplexed tandem PCR. The quality of diagnosis strongly depended on the molecular locus used. Nevertheless, many seeds were diagnosed down to species level, in concordance with their morphological identification, using one primer set from the core barcode site (<i>matK</i>). The number of specimens found in the Renaissance herbaria was not high; <i>Brassica nigra</i>, which is of great ethnobotanical importance, was the most common taxon. Thus, the combined use of independent means of species identification is particularly important when studying the early use of closely related crops, such as Brassicaceae.