Sex-determination methods are very diverse as they have become an enduring research field. Understanding the causes of gonadal development and elucidating the main factors involved in sex-determination of offspring required relating information from far-ranging areas such as cytology, embryology, morphology, molecular biology and even ecology and evolution. This article presents an overview of sex-determination in placental mammals, encompassing several levels of biological organization. The importance of the underlying molecular tools in the context of sex-determination assays and their implications in conservation genetics is also discussed.