Polyploid organisms pose substantial obstacles to genetic analysis, as molecular assay data are usually difficult to evaluate in a Mendelian framework. Green sturgeon (Acipenser medirostris) is a tetraploid species and is facing significant conservation challenges, including bycatch in ocean fisheries. We present here novel molecular genetic assays and analytical methodology for green sturgeon that allow discrimination of fish from the two visually indistinguishable distinct population segments (DPSs), and also provide individual-specific genetic tags. We show how the relative fluorescence intensity data from a standard quantitative PCR assay, designed for a biallelic single nucleotide polymorphism, can be grouped into “genotype categories” using standard analytical software and post-processing manipulation. We then show how these genotype category data can be used to discriminate green sturgeon from the southern DPS, which is protected under the US Endangered Species Act, and the northern DPS, which is not. We also show how these data can be used to reliably identify individual green sturgeon, and can therefore be used in capture/recapture analyses. Both types of identification are extremely accurate even when fewer than half of the assays are successfully called. We then apply these new techniques to show that proportions of the two green sturgeon DPSs are extremely different in the two major fishery areas where they are encountered as bycatch. While these assays and methods do not provide data that can be used in pedigree-based analyses, they are an important advance in the application of genetic analysis to conservation and management of polyploid organisms.