Objective In order to increase significantly the discriminatory potential of Y-STR systems available to the forensic community , We have developed and validated a four-color fluorescence multiplex PCR system consisting of 7 male-specific and polymorphic Y-STRs. Methods We designed three sets of constant primers chimerited into three groups of 7 Y-STR's specific primers and utilized fluorescence- labeled these three sets of constant primers to amplified simultaneously 7 Y-STRs in a reaction tube. Allele and haplotype frequencies at these Y-STRs were screened by ABI PRISM310 Genetic Analyzer and analyzed by Genotyper software. Results Following optimization of the polymerase chain reaction , DYS434, Y-GATA-A10,DYS531, DYS557 , DYS456 , DYS444 and DYS448 in a sample of 120 unrelated males , showed4, 5, 5, 8, 8, 6,7 alleles respectively. A total of 101 different haplotypes was identified,of which 89 (88. 11% ) were found in single individuals. The overall haplotypes diversity reached 0.9958. To the one case of mixture stains, our multiplex system drawn conforming conclusion comparing to the result of Y-STR genotypes in suspect's blood sample. Conclusion Our results show that the multiplex system of 7 Y-STR will be very powerful for establishing Y-STR database, the paternity testing and mixture stains identifying.