One of the most important requirements for cultivar identification in Vitis species is rapid and consistent genotyping using DNA-based markers. Genetic characterization and diversity of grapevine cultivars using molecular markers have been the subject of many studies. The Iranian grapevine biodiversity is one of the most important in the Middle East. In the present work, fourteen nuclear microsatellite markers were used to assess the genetic diversity and relationships among 25 local grapevine accessions collected from vine-growing areas of Qazvin province (Iran). A total of 81 distinct alleles were produced with amplified product sizes ranging from 91 to 432 bp. The number of observed alleles for each locus ranged from 2 (ISV3) to 9 (VVS3 and VVMD6), with the overall average of 5.79 allele per locus. The observed heterozygosity (Ho) ranged from 0.53 (ISV3) to 0.72 (VVSMD25), while the expected heterozygosity (He) varied between 0.49 (ISV3) and 0.87 (VVSMD25). The polymorphism information content (PIC) ranged between 0.50 for VVS3 and ISV3 to 0.87 for VVSMD25. However, Shannon’s information index (I) was found to be highest (2.13) in VVSMD25 locus, while the VVS3 locus had the lowest value with an average of 0.75 among SSR markers. According to Jaccard’s similarity coefficient and UPGMA algorithm, cluster analysis assigned the accessions into five groups. Despite similar names, the results revealed that the accessions belonged to different groups. The obtained PCoA scatter plots further strongly supported their UPGMA dendrogram results. Beside VVMD, VVS series of markers were also used in the present study and the results showed that these series of microsatellite markers were as efficient as VVMD series in terms of discrimination and polymorphism for studying grapevine cultivars. Among all the microsatellite loci, VVSMD25 (PID = 0.03, PIC = 0.87) was found to be the most informative among the accessions.