The current global agricultural challenges imply the need to generate new technologies and farming systems. In this context, rootstocks are an essential component in modern agriculture. Most currently used are those clonally propagated and there are several ongoing efforts to develop this type of plant material. Despite this tendency, lesser number of rootstock breeding programs exists in comparison to the large number of breeding programs for scion cultivars. In the case of rootstocks, traits evaluated in new selection lines are quite different: From the agronomic standpoint vigor is a key issue in order to establish high-density orchards. Other important agronomic traits include compatibility with a wide spectrum of cultivars from different species, good tolerance to root hypoxia, water use efficiency, aptitude to extract or exclude certain soil nutrients, and tolerance to soil or water salinity. Biotic stresses are also important: Resistance/tolerance to pests and diseases, such as nematodes, soil-borne fungi, crown gall, bacterial canker, and several virus, viroids, and phytoplasms. In this sense, the creation of new rootstocks at Centro de Estudios Avanzados en Fruticultura (CEAF) offers an alternative to stone fruit crop, particularly in Chile, where just a few alternatives are commercially available, and there are site-specific problems. The implementation of molecular markers in order to give support to the phenotypic evaluation of plant breeding has great potential assisting the selection of new genotypes of rootstocks. Marker-Assisted Selection (MAS) can shorten the time required to obtain new cultivars and can make the process more cost-effective than selection based exclusively on phenotype, but more basic research is needed to well understood the molecular and physiological mechanisms behind the studied trait.