The Mediterranean subpopulation of T. triunguis has been listed by IUCN as critically endangered (European Reptile and Amphibian Specialist Group 1996, category CR C2A). There are very few studies for determination of distribution and population genetics of this species in the world. A total of 13 polymorphic microsatellite loci were isolated from 42 samples of T. triunguis by using biotinylated (GT)(10), (CT)(10), (GATA)(5)-enriched library. The range of observed heterozygosities was 0.000-0.833 with a mean of 0.274, and the range of expected heterozygosities was 0.081-0.607 with a mean of 0.374. It was found that four loci deviated significantly from Hardy-Weinberg equilibrium after Bonferroni correction analysis. There was no evidence for large allele dropout. Null alleles may be present at ttmed-6, ttmed-8, and ttmed-9 loci, as suggested by the general excess of homozygote for most allele size classes. Designed microsatellite loci could provide a powerful tool for the population structure and conservation genetics studies of the Nile soft-shelled turtle and for captive breeding programs or to detect species illegal trade.