Sturgeons possess ZZ/ZW genetic sex determination system and are popular aquaculture target for high-priced black caviar originated from female roe. Therefore, the production of all-female seedling is highly desired, but this has not yet been achieved to date. In this study, we identified a short nucleotide sequence specific to putative Z and W scaffolds of Acipenser ruthenus genome and developed the PCR to discriminate Z and W alleles. Using this PCR, A. ruthenus (N = 1,280), A. baerii baerii (N = 66), and F2 of the bester hybrid (Huso huso × A. ruthenus: N = 31) were genotyped. Of those, the gonadal sexes were identified for 145 individuals, and all were concordant with their genetic sex. Additionally, we applied the Z/W-discriminative PCR for gynogenetic population of F3 bester and identified 25 out of 112 individuals (22.3%) showing only W amplicon, indicating that they are WW superfemales.