<p>North American <i>Vitis</i> species serve as a vital reservoir of genetic variation, offering valuable resources for molecular breeding programs focused on developing cultivars with enhanced resistance, adaptability, and quality traits for sustainable viticulture. The effective conservation and utilization of collections involving these species require a thorough understanding of their genetic diversity, population structure, and gene flow. In this study, 323 North American <i>Vitis</i> accessions categorized into six species groups were genotyped using 29 polymorphic microsatellite (SSR) markers. The objectives were to assess the genetic diversity and population structure, as well as to establish a representative core collection. Additionally, SSR markers associated with 15 resistance loci (<i>R</i>-loci) were analyzed to identify potential resistance to downy mildew, powdery mildew, black rot, Pierce’s disease, and phylloxera. The analysis revealed high genetic diversity, with 643 alleles identified, an expected heterozygosity (H_E) of 0.86, and an observed heterozygosity (H_O) of 0.74. Nine genetic groups were identified, with clear evidence of a substructure within some species. A core collection comprising 95 accessions capable of retaining all SSR alleles detected in the entire collection was established. Characterization of the <i>R</i>-loci revealed that 123 wild genotypes carried a single <i>R</i>-locus, 36 carried two <i>R</i>-loci, and 10 harbored three <i>R</i>-loci associated with pathogen resistance. The richness evident in the studied genetic pool represents an extensive reservoir of underexplored genetic diversity and crossbreeding potential. These findings have the potential to bolster the sustainable management, conservation, and subsequent molecular breeding applications of wild <i>Vitis</i> resources amidst emerging challenges in viticulture.</p>