New cultivars are result of the conservation and characterization of potato (Solanum) genetic resources in secondary germplasm banks. The objectives of this study were to assess phenotype diversity of 12 clones of 10 wild diploid potato species collection super series Rotata, and to determine their genetic diversity through simple sequence repeat (SSR) markers. Totally 63 alleles of 20 cpSSR loci were detected i.e. 3.15 alleles on average per one microsatellite locus. Alleles ranged from two to six per locus. The highest polymorphism was detected in the locus ntcp9 and lowest were recorded having by two alleles in seven of loci. The average value of observed heterozygosity (Ho) was 0.61, whereas the mean of polymorphic information contents (PIC) was 0.49. Intergenic regions had highest variability (Higr = 0.65) compare with introns (Hin = 0.54) and exons (Hex = 0.45) of the chloroplast genome. Molecular analyses were complemented with tuft morphological measurements according to the descriptor list for the genus Solanum. SSR-based markers highlight a tendency to separate two groups of Rotata wild diploids and show the possibility of duplicities of wild potato genetic resources in the current Czech in vitro collection.