In sturgeon aquaculture, all-female production is desirable due to the high value of caviar. Genetic sexing and the production of WW superfemales are important steps toward achieving this. In this study, we identified the WSR and ZSR primers for amplification of W- and Z-specific regions, respectively. WSR primers were designed on the gene W-linked RT RNase H-like domain containing protein (rnhW). The polymerase chain reaction (PCR) bands were obtained with the WSR primer only in phenotypic female sturgeons, indicating that stable genetic sexing was achieved in most species, including those captured around Hokkaido. Moreover, rnhW showed female-specific expression in the gonads during early sex differentiation in kaluga and Amur sturgeon. ZSR primers were developed from the orofacial cleft 1 candidate gene 1 protein homolog. Clear and distinct gel band patterns for ZZ, ZW, and WW genotypes were obtained using WSR and ZSR primers, consistent with genotypic estimations by quantitative PCR. This consistency confirmed the presence of WW superfemales among offspring produced by fertilizing ZW females with ZW pseudomales masculinized using 17α-methyltestosterone. Our findings provide new insights into the mechanisms of sex determination and differentiation in sturgeons, bringing the establishment of an all-female production system within reach.