In the present study, the genetic variations of 17 X-STR markers (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS6801, DXS7423, DXS6809, DXS6799, DXS7132, DXS9902, DXS6800, DXS6789, DXS10075, DXS10079, DXS6807, and DXS6803) were analyzed in 139 unrelated individuals in Nabeul, aiming to perform an X-STR database for anthropological and forensic purposes. Our results indicate that DXS6809 was the most polymorphic locus, whereas DXS6807 was the least informative marker. In addition, the obtained values for the statistical parameters of forensic interest, i.e., the power of discrimination in males (PDM) and females (PDF), as well as the mean exclusion chance in duos (MECD) and trios (MECT) have demonstrated that this panel of 17 X-STRs is highly informative and useful for forensic application and anthropological research. Additionally, pairwise genetic distances based on FST were calculated between Nabeul population and other populations extracted from the literature. Genetic distances were represented in a non-metric MDS plot and clustering of populations according to their geographic locations and their historical relationship was detected.