Hormone exposure in utero affects male- and female-typical behavior in animals, and these effects may persist in the next generation. Prenatal exposure to diethylstilbestrol (DES), a potent estrogen and endocrine disruptor, has been associated with a tendency toward greater heterosexual behavior in women, but the association in the next generation has not been studied. We evaluated the associations of maternal prenatal DES exposure with sexual behavior, sexual identity, and gender identity in 982 female offspring participating in the National Cancer Institute’s DES Third Generation Study, a cohort born to mothers who were prenatally exposed and unexposed to DES. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated from logistic regression models that included birth year. The ORs were 0.71 (CI 0.46–1.1) for DES in relation to non-heterosexual compared with heterosexual behavior, and 0.99 (CI 0.55–1.8) for non-heterosexual identity, compared with heterosexual identity. Results were similar after additional adjustment for education. Only three individuals reported a gender identity distinct from what was reported by the mother at cohort inception, preventing meaningful quantitative analysis of DES and gender identity. These data do not provide evidence of differences in sexual behavior and sexual identity in female offspring of mothers with and without prenatal exposure to DES.