Perennial kale is a rare leafy vegetable and forage crop that is mainly vegetatively propagated and therefore expensive to conserve ex situ. A genebank collection of 47 perennial kales and 34 reference samples from the main Brassica oleracea crop types were characterized with seven microsatellite markers in order to verify potential redundancies and to obtain more insight in the position of perennial kales within B. oleracea. Based on the obtained results and on data from previous studies, the collection was reduced with 49% to 24 perennial kales. Considering this level of reduction, it was estimated that the investments made for the final verification by microsatellite analysis are returned after only 4-year time. A principal coordinate plot clearly separated the perennial kales from the other crop types of B. oleracea, except in one case. This deviating accession of vegetatively preserved perennial kale clustered closely together with the single seed-preserved accession of perennial kale included in the study. These two accessions occupied an intermediate position between the group of vegetatively propagated perennial kales and the group of seed-propagated Brassica accessions, suggesting a hybridization background with another B. oleracea crop type. The microsatellite study demonstrated a close genetic relationship among the investigated perennial kales and their unique position within B. oleracea.