The determination of the sex in turtles and tortoises, that do not show an evident sexual dimorphism, is an increasingly required practice of the Veterinarian in unconventional animals. These procedures are applied in numerous fields: from amateur breeding for commercial purposes, to the prevention of reproductive diseases (formation of reproductive groups, prevention of unwanted births) and for conservation purposes (repopulation plans of threatened species). These objectives see the need to develop minimally invasive techniques useful for determining the sex of the specimens in a quick, safe and painless way.