Quantifying primary sex ratios is necessary for studies in a wide range of areas including adaptive sex ratio modification, population demography, and sex-biased developmental mortality. Adult and penultimate male spiders are easy to sex, due to the great thickening of the male pedipalps, which are used for delivering sperm to the female reproductive tract. However, in many spider species, males and females are apparently monomorphic at hatching, are difficult to rear, and cannibalize their siblings, making assessment of primary sex ratios problematic. One technique for sexing spiders is karyotyping, but this can be challenging and time-consuming, particularly for species with high fecundity, and often requires destructive sampling. Here we report that, although apparently monomorphic, early-instar juveniles of two species of black widow spiders (Latrodectus hasselti Thorell 1870 and Latrodectus hesperus Chamberlin & Ivie 1935) can be sexed reliably. Palp width measurements are significantly different for males and females at the 3rd instar, with the palpi of juvenile females thinner than those of males. Moreover, sex identification with 89–100% accuracy can be achieved by an experienced observer visually inspecting the palpi of 3rd instar spiderlings under a dissecting microscope. Our results suggest that minimal investment in a pilot study can yield an accurate method for sexing juvenile spiders in the laboratory or field. The suitability of this method should be examined in other species with apparently monomorphic spiderlings, particularly those in which adult males have significantly enlarged palpi.