We studied plastron-shaped asymmetry of the Eurasian Spur-Thighed Tortoise, Testudo graeca ibera, as related to life stage, gender, and distribution. Our analyses of 523 individuals showed that fluctuating asymmetry (FA) in plastron shape varied with gender (males exhibited higher levels of FA than did females) and across populations, whereas life stage had no significant effect. Although we could not identify the potential sources of variation responsible for the observed patterns of developmental instability, our study shows the value of FA as a method for studies of developmental instability in turtles.