The evaluation of ‘ex situ’ germplasm collections allows us to use and conserve them more efficiently. A collection of 107 durum wheat (Triticum durum Desf.) accessions collected in Southern Italy from 1947 to 2009 was characterized using six morphological traits and 30 SSR markers. On the basis of the plant height, these accessions were classified into Pop 1 (plant height ≤70 cm), Pop 2 (70 cm <plant height <100 cm) and Pop 3 (plant height ≥100 cm). Significant differences were observed for morphological traits in these populations confirming the effects of the introduction of varieties with dwarfing genes. The 30 SSR markers identified 115 alleles, with an average of 3.83. The estimates of Na, Ne, I and He for each population suggested a decrease in genetic diversity after the introduction of dwarfing genes in durum wheat germplasm. The level and the distribution of genetic diversity in the materials analysed in this study could be used as genetic criteria to protect these accessions in ex situ collections, to further investigate their characteristics and to use them both for breeding systems and for the improvement of typical products.