Grapevine (Vitis vinifera subsp. vinifera) is one of the most widespread and economically important perennial fruit crops in the world. Viticulture has changed over the years in response to changing environmental conditions and market demands, triggering the development of new and improved varieties to ensure the crop’s sustainability. The aim of this review is to provide a perspective on the recent developments in biotechnology and molecular biology and to establish the potential of these technologies for the genetic improvement of grapevine. The following aspects are discussed: (i) the importance of molecular marker-based methods for proper cultivar identification and how NGS-based high-throughput technologies have greatly benefited the development of genotyping techniques, trait mapping, and genomic selection; (ii) the recent advances in grapevine regeneration, genetic transformation, and genome editing, such as new breeding technology approaches for enhanced grapevine yield, quality improvement, and the selection of valuable varieties and cultivars. The specific problems and challenges linked to grapevine biotechnology, along with the importance of integrating classical and new technologies, are highlighted.