Bread wheat (Triticum aestivum L.) is an economically and nutritionally important cereal crop in the Mediterranean region. Characterization of wheat germplasm by means of DNA fingerprinting techniques provides a tool to assess genetic diversity and to identify varieties. In this study, six Tunisian bread wheat cultivars were characterized by AFLP and SSR markers. Five AFLP primer pairs showed clear different patterns and seems to be the most suitable for analysis of the bread wheat varieties. Three SSR primers were polymorphic with more than two alleles. The pairwise genetic similarities (GS) based on these molecular markers were calculated and used to construct a dendrogram that allowed the discrimination of the six cultivars. The GS among the six varieties ranged from 0.79 to 0.36. The six varieties used in this work clustered into four groups using either AFLP or SSR markers. A high GS was found between Tebecca and Vaga Varieties which have a similar pedigree.