Currently in physical anthropology there is a need for reliable methods of sex estimation for immature individuals and highly fragmented remains. This study develops a sex estimation technique from discriminant function analysis of the bony labyrinth as it matures before puberty and can survive taphonomic conditions that would destroy most other skeletal material. The bony labyrinth contains the organs of hearing and balance. For this reason biologists and paleoanthropologists have undertaken research in this area to understand evolutionary changes in locomotion. Prior studies have found clear differences between species, but within-species variation has not been satisfactorily investigated. 3D segmentations of the left and right labyrinths of 94 individuals from a Cretan collection were generated and measured. Mean measurements of height, width, size, and shape indices were analyzed for sexual dimorphism, bilateral asymmetry, and measurement error. Significant sexual dimorphism was detected for several measurements. For sex estimation, the single best variable was the radius of curvature of the posterior semicircular canal, which achieved 76% accuracy. Two multivariate functions increased accuracy to 84%. Although these equations are less accurate than equations for complete long bones and crania, they appear to be as accurate as or better than other techniques for sexing immature individuals and temporal bones. Am J Phys Anthropol 151:290–301, 2013. © 2013 Wiley Periodicals, Inc.