Kale (Brassica oleracea L. var. acephala) is a widely appreciated vegetable with a century-old history of cultivation in Italy. The present study was addressed to the collection and characterization of kale germplasm traditionally cultivated in Apulia, Southern Italy, nowadays at risk of genetic erosion. In total, nineteen Apulian kale accessions were acquired. Genotyping by means of simple sequence repeat (SSR) DNA markers led to the identification of highly informative primer combinations and highlighted significant patterns of molecular variation among accessions. Consistently, significant differences were observed with respect to morpho-agronomic traits, including yield and harvesting time, and the content of bioactive compounds, namely total phenols, flavonoids, and anthocyanins, associated with antioxidant activity. Overall, this study led to the establishment of an ex situ collection of great importance to preserve endangered Apulian kale germplasm and to provide seed access to potential growers. Meanwhile, it offers a first characterization of Apulian kale, useful to promote its consumption and valorisation through breeding programmes.