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Andalusia is a Spanish region that is home to numerous minority varieties due to its diversity and territorial extension, offering the local viticulture the possibility of diversifying its wine production. The genotypic characterization of 98 specimens from six areas with a winemaking tradition in Andalusia was carried out between the years 2020 and 2022, by means of thirteen microsatellite markers, including the nine recommended by the OIV. A total of 33 different genotypes were obtained, 20 of which corresponded to profiles of already described varieties (11 of them are of 6 minority cultivars in Andalusia: ‘Rojal Tinto’, ‘Beba’, ‘Zurieles’, ‘Rome’, ‘Hebén’, ‘Mollar Cano’, ‘Listán Prieto’, ‘Listán del Condado’, ‘Jarrosuelto’, ‘Negra Dorada’, and ‘Mantúo de Pilas’), while the other 12 profiles did not match with previously identified varieties. These profiles were registered in the database of the IFAPA “Rancho de la Merced” Germplasm Bank. The eco-geographical groups of the new identified genotypes were determined through an analysis of genetic diversity. The presence of grapevine fanleaf virus, grapevine fleck virus, and grapevine leafroll-associated viruses was also determined due to the requirement of healthy clones of the new varieties for their potential interest to be authorized for cultivation in Spain.