A large repository of 441 rootstocks and other non-vinifera grape cultivars was established, beginning in 2013, by the University of Milan with the aim of collecting most of the genetic variability of Vitis species useful for rootstock genetic improvement. This kind of collection is the largest in Italy, with accessions from different donor institutes in California, Spain and Italy, and also encompassing progenies obtained by University of Milan breeding programs. A preliminary genetic characterization was performed, as is usually done for better management of grape germplasm. Eighty-six per cent of the accessions were evaluated by genotyping with simple sequence repeat (SSR) markers and also by phenotyping the sex of flowers. Twenty-two carefully selected and highly polymorphic SSR markers were used, out of 41 taken into consideration, which encompassed the nine markers applied internationally for V. vinifera genotyping and 26 VChr SSRs having a longer core tandem repeat. However, five of the 22 markers were shown to be critical when applied on a large scale for genotyping of non-vinifera plants and were subsequently discarded. SSR profile comparisons showed that more than a third of the accessions were redundant. Fewer than half were identified as true-to-type, and more than a third remained anonymous. These anonymous, as-yet uncharacterized genotypes represent a precious reservoir of genetic diversity. Analysis of the genetic structure revealed four main groups, with more than a third of genotypes classified as admixed. A genetic core collection able to capture the whole allelic variation was set up as a valuable starting point for both rootstock breeding and genome-wide association studies.