The X-chromosome has valuable characteristics for population genetic studies. In order to investigate the genetics of the human Mediterranean populations further, we developed a 25 X-chromosome SNP-multiplex typing system. The system was based on PCR multiplex amplification and subsequent multiplex single base extension with the SNaPshot reaction, capillary electrophoresis and multicolor fluorescence detection. We investigated 11 Mediterranean populations with the 25 X-chromosome SNPs. A high overall homogeneity was found among the Mediterranean populations except for Moroccans, who differed genetically from the rest of the populations in the Mediterranean area. This result supports the hypothesis of a low incidence of the south-north genetic interchange at the western shores of the Mediterranean basin. A low genetic distance was found between populations in the Middle East and the western part of the Mediterranean area most likely reflecting the strong effect of the Neolithic wave. A certain level of background linkage disequilibrium among the 25 SNPs on the X-chromosome in Ibiza and Cosenza was observed, possibly as a consequence of their demographic history