Within the framework of the program of rehabilitation and conservation of the Tunisian wine heritage and considering the importance of the variability of the local grape varieties, morphological and molecular microsatellite markers were used to list 61 cultivated autochthonous Tunisian grape varieties. The use of 17 ampelographic descriptors allowed to obtain a high number of morphotypes and to discriminate all cultivars. The multi-variety analyses Type PCA showed that only 6 morphological variables among 17 participate in the structuring of the different grape varieties. The color variable represented by the characters OIV 015, OIV 015-2 and OIV 051 constitutes almost all the structuring supported by the second axis of the PCA. Furthermore, the polymorphism revealed using 15 microsatellite loci resulted in a very high allele number (121) and a very high heterozygosity (0.76). For the VVMD7, SCU04, VVMD17 and VVMD14 loci, the heterozygosity was underestimated. Therefore, extreme caution should be considered when using these primers.