Chloroplast DNA restriction site variation for 17 endonucleases was surveyed for the large single-copy region of the genome in 26 taxa of the genus Diplotaxis and compared with previously mapped site mutations in other members of the Subtribe Brassicinae (Tribe Brassiceae, Cruciferae). A total of 259 restriction site and length mutations were observed, 206 (80%) of which showed variation at the interspecific level. Phylogenetic analysis indicated a clear division of the genus Diplotaxis into the same two lineages for the Subtribe Brassicinae, described previously by Warwick and Black as Rapa/ Oleracea and Nigra. Levels of genetic divergence and taxon groupings suggested by the restriction site variation were highly consistent with previously recognized cytodemes or crossing groups and the geographical distribution of Diplotaxis taxa. However, the data were inconsistent with the morphologically based taxonomic delimitation of the genus, certain subgeneric circumscriptions, and even species delimitation in the case of D. virgata. Diplotaxis species were separated into three major groups in each of the two lineages. In the Rapa/ Oleracea lineage, Group A included D. erucoides ssp. erucoides and D. cossoniana. Group B included two subgroups: B (i) D. tenuifolia, D. cretacea, and D. simplex and B (ii) D. harra. Group C included D. viminea and D. muralis. Within the Nigra lineage, group D included D. siettiana, D. ibicensis, D. brevisiliqua, and D. gomezcampoi. Group E included D. brachycarpa. Group F included three subgroups: F (i) D. assurgens, D. tenuisiliqua, an accession of D. virgata from southern Morocco (DVA), and D. siifolia; F (ii) D. berthautii and D. virgata f. sahariensis; and F (iii) D. catholica, D. catholica var. rivulorum, D. virgata ssp. virgata. These groups often showed greater genetic closeness to other species from other genera in the Subtribe than to other species of Diplotaxis.