The isolation and characterization of six polymorphic loci from a Testudo hermanni genomic library is reported. Enrichment was performed for AC but four of the characterized microsatellites present also an additional motif. Variability was tested on populations of the two recognized subspecies, Testudo hermanni hermanni and Testudo hermanni boettgeri. For one locus, a size range specific for the subspecies T. h. hermanni was observed. These are the first primers identified directly in the genome of T. hermanni.