X-chromosomal short tandem repeats (X-STRs) loci are used for forensic practice in recent years which play increasingly important roles in some complex kinship cases. In this paper, a new multiplex polymerase chain reaction (PCR) system which can simultaneously analyze 11 X-STR markers (DXS8378, DXS6795, DXS7132, DXS6803, DXS9898, DXS6801, DXS7133, GATA165B12, HPRTB, DXS8377 and DXS7423) was developed. The samples of 1,605 (742 males and 863 females) unrelated individuals from Tibetan and Northern Han population were successfully analyzed using this multiplex system. A total of 103 alleles for all the loci were observed. Hardy-Weinberg equilibrium tests demonstrated no significant deviation from expected values (P > 0.05) for all of the 11 X-STR loci in the two studied populations. Polymorphism information contents of the loci were 0.3864-0.9013, and powers of discrimination in females of the loci were 0.6317-0.9845. There were no statistically significant differences between Tibetan and Northern Han populations in allele distribution of the 11 X-STR loci, in line with analysis of molecular variance (AMOVA) results. Our work indicates that this multiplex system is useful for forensic analysis for the two populations in China.