This study tested the hypothesis that steroid hormone metabolites can be measured in anuran feces and their concentrations used to identify the sex of adults. Fecal samples from American toads, Bufo americanus, and boreal toads, B. boreas boreas, were extracted using ethyl acetate, and the concentrations of estradiol, progesterone and testosterone metabolites were measured by enzyme immunoassays with antibodies commonly used to evaluate steroid hormone concentrations in mammalian species. In American toads, mean testosterone metabolite concentrations (P<0.05) between males (224.3±15.5 ng/g feces) and females (80.7±10.6 ng/g), but estradiol and progesterone metabolite concentrations did not. In contrast, estradiol immunoreactivity differed (P<0.05) between male (19.0±1.8 ng/g) and female (48.3±6.3 ng/g) boreal toads. Progesterone and testosterone metabolite concentrations did not differ. Fecal hormone metabolite analysis offers a promising noninvasive approach to gender identification in anuran amphibians. However, the group of metabolites differentiating gender may not be consistent among species. Zoo Biol 0:1–12, 2005. © 2005 Wiley-Liss, Inc.