The genetic variability of the tadpole shrimp Triops cancriformis was studied through the analysis of 12 nuclear microsatellite loci. Seven of these loci were newly obtained from an enriched genomic library using a trinucleotide biotinylated probe (CAG)11. The study was conducted on six populations that previous genetic studies demonstrated to be poorly differentiated, notwithstanding their different reproductive strategies (gonochorism, parthenogenesis, androdioecy). The newly isolated microsatellite loci showed a higher genetic variability than those previously isolated, probably in relation with their different motif structure. Different patterns of variability were scored at both locus and population levels, the latter probably being caused by the different reproductive modalities. Wright's F‐statistics, AMOVA, and ISOLDE analyses showed that populations are well structured and isolated, again as a result of different reproductive modalities, but also of geographic distances and low migration flow.