Sex estimation from the skull is commonly performed by physical and forensic anthropologists using a five-trait scoring system developed by Walker. Despite the popularity of this method, validation studies evaluating its accuracy across a variety of samples are lacking. Furthermore, it remains unclear what other intrinsic or extrinsic variables are related to the expression of these traits. In this study, cranial trait scores and postcranial measurements were collected from four diverse population groups (U.S. Whites, U.S. Blacks, medieval Nubians, and Arikara Native Americans) following Walker's protocols (total n = 499). Univariate and multivariate analyses were utilized to evaluate the accuracy of these traits in sex estimation, and to test for the effects of population, age, and body size on trait expressions. Results revealed significant effects of population on all trait scores. Sample-specific correct sex classification rates ranged from 74% to 94%, with an overall accuracy of 85% for the pooled sample. Classification performance varied among the traits (best for glabella and mastoid scores and worst for nuchal scores). Furthermore, correlations between traits were weak or nonsignificant, suggesting that different factors may influence individual traits. Some traits displayed correlations with age and/or postcranial size that were significant but weak, and within-population analyses did not reveal any consistent relationships between these traits across all groups. These results indicate that neither age nor body size plays a large role in trait expression, and thus does not need to be incorporated into sex estimation methods. Am J Phys Anthropol 154:259–269, 2014. © 2014 Wiley Periodicals, Inc.