Many X-chromosome short tandem repeats (X-STRs) have been validated for forensic use even if further studies are needed on allele frequencies and mutation rates to evaluate the extent of polymorphism in different populations and to establish reference databases useful for forensic applications and for anthropological studies. A single multiplex reaction of seven X-STRs, which includes the DXS6789, HUMARA, DXS10011, DXS7423, HPRTB, DXS6807, DXS101 loci, is presented and their allele frequency distribution in a large population sample including 556 subjects (268 females and 288 males) analysed by five forensic laboratories of Central and Northern Italy is shown. Our results demonstrate the feasibility of a single amplification/detection reaction involving seven markers of the X chromosome, which can be fruitfully used in complex kinship analysis.