Microsatellite markers are a valuable tool to facilitate the management of germplasm collections and assess genetic diversity. This study reports the genetic characterization of a large collection of 379 rootstocks and other non-vinifera accessions maintained at the University of Milan, Italy. Accessions were genotyped with 22 highly polymorphic microsatellite markers, including the nine “international” loci used for grapevine identification, three VMC, three VrZAG, and seven VChr loci; 17 loci were retained to identify cultivars, investigate genetic diversity, analyze pedigrees, infer population structure, and design a core collection. This study identified 232 unique genotypes; the allelic profiles of 70 rootstocks were confirmed according to the literature and databases, while the profiles of 43 rootstocks were proposed for the first time. Pedigree analysis highlighted 77 parents-offspring trios and 44 parent-offspring relationships, some of them already known and others new. Genetic-structure analysis showed a more likely number of three ancestral groups, with a high percentage of admixed samples. A structure based on the genetic background of genotypes was not observed. A core collection of 70 genotypes captured 100% of the entire number (373) of detected alleles. Most of these genotypes were unidentified or poorly characterized. The information provided in this paper could assist breeders in their efforts to exploit still unexplored individuals useful for long-term breeding plans.