Fifty-nine Brassica oleracea cultivars, belonging to five botanical varieties, were evaluated for microsatellite (SSR) polymorphisms using 11 database sequence derived primer pairs. The cultivars represented 12 broccoli (Brassica oleracea var. italica), ten Brussels sprouts (B. o. var. gemmifera), 21 cabbage (B. o. var. capitata, including the groups white and red cabbage), six savoy cabbage (B. o. var. sabauda), and ten cauliflower (B. o. var. botrytis) cultivars from 13 seed suppliers. The 11 primer pairs amplified in total 47 fragments, and differentiated 51 of the cultivars, whereas the remaining eight cultivars were differentiated from the rest in four inseparable pairs. All SSR markers, except one, produced a polymorphic information content (PIC value) of 0.5 or above. The average diversity for all markers within the tested material was 0.64. There was no major difference in the diversity within botanical varieties and groups. The cluster analysis and the resulting dendrogram showed that the cultivars tended to group within these taxonomic units. The present study substantiates the use of microsatellite markers as a powerful tool for cultivar differentiation and identification in vegetable brassicas.