Handedness preference was assessed in 205 boys with gender identity disorder and 205 clinical control boys referred for other reasons. Boys with gender identity disorder were significantly more likely to be left-handed than the clinical control boys (19.5% vs. 8.3%, respectively). The boys with gender identity disorder, but not the clinical control boys, also had a significantly higher rate of left-handedness compared to three independent, general population studies of nonreferred boys (11.8%; N = 14,253) by Hardyck, Goldman, and Petrinovich (1975), Calnan and Richardson (1976), and Eaton, Chipperfield, Ritchot, and Kostiuk (1996). Left-handedness appears to be a behavioral marker of an underlying neurobiological process associated with gender identity disorder in boys.