To accurately infer population structuring and manage species, it is advised to combine data obtained from mitochondrial DNA (mtDNA) with data from fast evolving markers such as microsatellites. To date, the evolutionary history of a threatened Mediterranean species, Mauremys leprosa, was inferred based solely on mtDNA data, which may lead to an incomplete, or partially explained, population structuring. We tested the cross-amplification of 16 microsatellite loci in 190 individuals of M. leprosa from six Iberian and two African populations. We obtained a successful set of 11 polymorphic loci with 2-18 alleles and observed heterozygosity ranging from 0.007-0.783. This panel of loci can be used for future research in M. leprosa, such as population structuring, analysis of gene flow in secondary contact zones, paternity analyses, changes in phenotypic traits and to assemble a comprehensive genetic dataset (mtDNA and nuDNA) that will allow the geographic assignment of individuals of unknown origin. These tools will help managing M. leprosa populations throughout the species’ range.