Waterhemp [Amaranthus tuberculatus (Moq.) J. D. Sauer] and Palmer amaranth (Amaranthus palmeri S. Watson) are troublesome weeds of row-crop production in the United States. Their dioecious reproductive systems ensure outcrossing, facilitating rapid evolution and distribution of resistances to multiple herbicides. Little is known, however, about the genetic basis of dioecy in Amaranthus species. In this work, we use restriction site–associated DNA sequencing (RAD-Seq) to investigate the genetic basis of sex determination in A. tuberculatus and A. palmeri. For each species, approximately 200 plants of each sex were sampled and used to create RAD-Seq libraries. The resulting libraries were separately bar-coded and then pooled for sequencing with the Illumina platform, yielding millions of 64-bp reads. These reads were analyzed to identify sex-specific and sex-biased sequences. We identified 345 male-specific sequences from the A. palmeri data set and 2,754 male-specific sequences in A. tuberculatus. An unexpected 723 female-specific sequences were identified in a subset of the A. tuberculatus females; subsequent research, however, indicated female specificity of these markers was limited to the population from which they were identified. Primer sets designed to specifically amplify male-specific sequences were tested for accuracy on multiple, geographically distinct populations of A. tuberculatus and A. palmeri, as well as other Amaranthus species. Two primer sets for A. palmeri and four primer sets for A. tuberculatus were each able to distinguish between male and female plants with at least 95% accuracy. In the near term, sex-specific markers will be useful to the A. tuberculatus and A. palmeri research communities (e.g., to predict sex for crossing experiments). In the long-term, this research will provide the foundational tools for detailed investigations into the molecular biology and evolution of dioecy in weedy Amaranthus species.