Different biotechnological tools for Prunus breeding including alternative germplasm souces, new evaluation techniques, and molecular markers development, are described. In germplasm improvement, the introduction of genes from related Prunus species confering several traits are being pursued. On the other hand, twin seeds (two embryos within the same seedcoat) have produced seedlings useful for genetic studies. Promising propagation methods include in-vitro techniques for the evaluation of plant material, allowing the early evaluation of a high number of genotypes. In addition, the growth of seedlings in controlled environments, including the induction of an artificial rest period in cold chambers, provides a useful strategy for obtaining vigorously growing plants year round. Molecular markers have also become an essential tool in Prunus breeding studies. Different types of molecular markers have been employed for the genetic characterization of germplasm, the establishment of genetic relationships between cultivars and species, and the construction of genetic maps. Methodologies for the analysis of marker-assisted selection include the use of mapping populations segregating for desired characters and bulk segregant analysis. To date, molecular markers associated to 19 qualitative traits (monogenic u oligogenic) and 18 quantitative traits (polygenic) have been described in different Prunus species.