The characterization of demographic features in natural populations is important to understand recruitment dynamics, mating systems, and propensities for loss of genetic diversity. The accurate identification of sex can be difficult in sexually monomorphic species. We present a melt curve analysis based on qPCR assays for lake sturgeon (Acipenser fulvescens) sex identification and validation using the AllWSex2 marker and adult males and females (total N = 143) with an assigned field sex from two populations (Black Lake, MI, and Menominee River, MI–WI border, USA). The assay is highly accurate (~98% concordance between putative or confirmed field and molecular sex classification), fast, and easy to use. The detected inconsistencies between field and molecular sexing were partially attributed to field-sexing uncertainty. This method will allow researchers to rapidly determine the sex of individuals of this regionally threatened species to use in conservation efforts and research.