A fundamental biological question that has puzzled, but also fascinated mankind since antiquity is the one pertaining to the differences between sexes. Ancient cultures and mythologies poetically intended to explain the origin of the two sexes; philosophy offered insightful albeit occasionally paradoxical perceptions about men and women; and society as a whole put forward numerous intuitive observations about the traits that distinguish the two sexes. However, it was only through meticulous scientific research that began in the 16th century, and gradual technical improvements that followed over the next centuries, that the study of sex determination bore fruit. Here, we present a brief history of sex determination studies from ancient times until today, by selectively interviewing some of the milestones in the field. We complete our review by outlining some yet unanswered questions and proposing future experimental directions.