The existence of numerous grapevine varieties has led to the emergence of many cultivars that are often poorly identified or known by different names in various regions. Currently, molecular biology methods that require high-quality genomic DNA allow for the genetic identification of these different varieties. Our study is considered a first step toward the genetic identification of grapevines in Algeria and the optimization of a protocol for better nucleic acid extraction. This development was carried out on 20 grapevine cultivars sampled from the El-Kantara commune, in the province of Biskra, Algeria. We optimized a cost-effective CTAB-based protocol and developed a simple method for preparing leaf samples as a preserved powder, which can be used at any time to obtain intact genomic DNA. The average concentration, yield (2385 µg/g), and purity of the genomic DNA extracted using the optimized protocol are very satisfactory compared to the reference protocol.