Ninety-five microsatellite loci were isolated from the output of two Next Generation Sequencing projects: a genomic Illumina RADSeq and a transcriptomic Roche 454 cDNA sequencing of the Adriatic sturgeon (Acipenser naccarii). From a total of 7697 loci identified, 72 were characterized and 57 were polymorphic in the tetraploid A. naccarii, providing new markers suitable for parental allocations to be performed for future conservation actions. Loci isolated from the transcriptome showed a higher level of polymorphism, which could be related to the different sequencing approaches used. Notably, a higher rate of duplication was observed in the transcribed loci, pointing to a preferential retention of sequences with a functional role after whole genome duplications occurred in sturgeons. Cross-species amplification of these markers was also assessed in the diploid beluga sturgeon (Huso huso), with the aim of setting up a panel of markers to be applied in future conservation programmes regarding this species.