The steadily increasing world population and the concurrent reduction in cultivated lands are two major threats to food security, especially in the underdeveloped countries. Among the many strategies that can mitigate this situation modern biotechnologies play a central role. Many tools are available to scientists to face the challenges of increased production needs and sustainable agriculture. Among these, genetic modification and molecular breeding appear to be the most promising. Both of these approaches require the combined use of technologies such as genome sequencing, genotyping and phenotyping and large-scale data analysis and mining to determine genes and functions amenable to manipulation in the target species. Molecular markers, used in both target gene studies and assisted breeding, represent a powerful tool. This chapter deals with their nature and applications, and also describes some case studies where their use in marker-assisted selection has positively affected some target crop species.