Eruca vesicaria subsp. sativa (syn. E. sativa) is a cruciferous vegetable and oilseed crop that is high in erucic acid. It occurs throughout the Mediterranean region and western Asia, and has been naturalized elsewhere as a crop/weed escape. It is closely related to subsp. vesicaria and subsp. pinnatifida, which are endemic to Spain and north-western Africa, respectively. This study evaluated patterns and levels of diversity in the three subspecies based on 234 amplified fragment length polymorphisms (AFLP), and evaluated agronomic and seed quality data in a field trial in western Canada. AFLP data revealed three main clusters: ‘Sativa’ (33 accessions of subsp. sativa), ‘Vesicaria’ (nine accessions of subsp. vesicaria) and a ‘Pinnatifida’ cluster (one accession of subsp. pinnatifida and three Moroccan accessions of subsp. sativa). The Sativa cluster separated into Mediterranean and Asian groups, likely reflecting differences in origin (wild versus cultivated) or primary usage, vegetable versus seed oil. The origin of the introduced Mexican population was confirmed as subsp. sativa. The highest levels of diversity were found in the Sativa cluster (88% AFLP polymorphisms) and the least in the Vesicaria (56%) and Pinnatifida (39%) clusters. Extensive variation was observed among the 159 subsp. sativa accessions evaluated in the field trial, and overall findings indicated a favourable agronomic potential.