Common DNA-based sexing assays have been widely used for the conservation and management of mammals and birds. However, many fishes do not have genetic sex determination and in those that do, the plasticity of the genes involved means that species-specific assays are normally required. Such DNA-sexing markers would be especially valuable in lake sturgeon (Acipenser fulvescens) because of their sexual monomorphism, delayed sexual maturity, and conservation status. We tried to identify genetic differences between male and female lake sturgeon using several different molecular genetic methods, including randomly amplified polymorphic DNA, representational difference analyses, subtractive hybridization, and a candidate gene approach. Ultimately, a number of genes were identified but none was sex-specific. Although the ultimate mechanism of sex determination is yet unknown, it is possible that sex determination is environmental in lake sturgeon, especially since recent studies have also failed to identify sex determination genes in other sturgeon species.