Order Acipenseriformes (sturgeons and paddlefishes) is an ancient lineage of osteichthyan fishes (>200 million years old) with most extant species at conservation risk. A relatively basal species, the pallid sturgeon, Scaphirhynchus albus, is a federally endangered species native to the Mississippi and Missouri River basins. Hybridization with sympatric shovelnose sturgeon, S. platorynchus, is one of several threats to pallid sturgeon. Current molecular markers cannot reliably distinguish among pure species and multigenerational backcrosses. This information is critical for implementation of management strategies to increase populations through natural reproduction and artificial propagation. Genotypes from a large panel of unlinked single-nucleotide polymorphisms (SNPs) may provide greater resolution of the two species; however, paralogous sequence variants (PSVs) within individuals resulting from an ancient whole genome duplication event confound SNP development. The aim of this study was to produce pallid sturgeon gynogens that contain 100% homozygous DNA contributed by only the maternal parent and have enough DNA for future SNP marker development. When homozygous gynogens are sequenced, heterozygosity at a locus within an individual indicates the presence of incorrectly aligned sequences that contain PSVs; accurate identification of these multi-locus contigs can facilitate their exclusion when developing disomic markers. In this study, we attempted to produce two types of pallid sturgeon gynogens: a) haploid gynogens produced from the activation of pallid sturgeon eggs with ultraviolet-irradiated sperm from the distantly related paddlefish (Polyodon spathula), and b) doubled haploids produced from the activation of pallid sturgeon eggs with irradiated paddlefish milt followed by thermal shock to suppress the first mitotic division. Production of doubled haploids, gynogens with 100% homozygous DNA and double the genome content of haploid gynogens, was pursued because it was originally unknown if haploid gyongens would survive long enough to attain enough genetic material for SNP marker development. We performed flow cytometry and microsatellite genotyping on the specimens in order to confirm haploid and doubled haploid status. Our study was unable to successfully yield doubled haploids; however, we successfully produced haploid gynogens that contained enough nuclear DNA for our future SNP marker development study. Interestingly, this study also produced paddlefish × pallid sturgeon hybrids in the control groups in two separate years; this is the first study to report viable offspring between the paddlefish and a Scaphirhynchus sturgeon species and reflects on the malleability of the genomes of the species in this order.