The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.