Acipenser dabryanus is listed as a Critical Endangered species in the IUCN Red List and the first class protected animals in China. Fortunately, A. dabryanus specimens are being successfully bred in captivity for conservation. However, for effective ex situ conservation, we should be aware of the genetic diversity and the degree of relatedness of the individuals selected for breeding. In this study, we aimed at the development of novel and reliable microsatellites used for the genetic study of A. dabryanus. A total of 14,321 simple sequence repeats (SSRs) were detected by transcriptome sequencing and screening. We selected 20 novel and polymorphic microsatellites (non-dinucleotide) with good repeatability from the 100 tested loci for a subsequent genetic and paternity study. A set of captive broodstock (F1 stock, n = 43) and their offspring (F2 stock, n = 96) were used to examine the efficiency of the 20 SSRs for assigning parentage to offspring, with an allocation success of 91.7%. We also found that only a few families predominantly contributed to the progeny produced by the 43 breeders. In addition, mitochondrial DNA data showed that the captive broodstock (F1 individuals) had an excellent probability of the same lineage, implying that a high level of inbreeding may have occurred in these individuals. Our research provides useful information on genetic diversity and reproductive pattern of A. dabryanus, and the 20 SSRs developed in this study can be applied to the future breeding program to avoid inbreeding for this stock or other related species of Acipenseriformes.