Genomic DNA was extracted from whole blood of 198 unrelated health individuals of Tujia ethnic group from south China's Hunan Province. Genotyping and detection of PCR products were carried out on denaturing polycrylamide gel electrophoresis followed by silver staining. Allele frequencies and genotype frequencies were computed. Deviation from Hardy-Weinberg equilibrium and differences of gene distribution were examined by SPSS 13.0. Fixation index, genetic polymorphism and indices of forensic application were calculated using Fstat and Powerstats, respectively. The results revealed that the frequencies of 65 alleles were distributed from 0.0048 to 0.6170. Among the ten X-STR loci, DXS6789, DXS6799 and HPRTB presented lower diversity and differentiation, while DXS7133 and DXS7423 showed lower value in forensic application. Results of multiple comparisons by loci showed that difference between German, Italian and Tujia population were the most dominant, and it suggested that great genetic differences did exist between Caucasian and Mongo-lian. In conclusion, DXS6804, DXS7132, DXS7130, DXS8378, DXS6789, DXS6799, DXS7424 and HPRTB had a good value in forensic identification, paternity testing of female and disease related study for Tujia population.