In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.