Y chromosome-specific short tandem repeat (Y-STR) analysis has become a widely accepted tool for forensic DNA analysis. The PowerPlex Y System is a fluorescent multiplex PCR kit that enables simultaneous amplification of 12 Y-STR loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439). In this study, we evaluated the application of this kit to forensic evidential samples.   The complete profiles were successfully determined using the recommended condition of 30 PCR cycles in the modified 12.5 μL reaction volumes. Using this protocol, it was possible to type all Y-STR loci from 62.5 pg of male DNA. The male/male DNA mixtures produced full profiles from the minor component up to the 1: 2.5 ratio, which contained 0.2 ng of minor male DNA and 0.5 ng of major male DNA. The minor male component in a male/female DNA mixture was detected in all loci up to the 1:3200 ratio, which contained 125 pg of male DNA and 400 ng of female DNA. Experimentally decomposed DNA samples produced full Y-STR profiles using 10 ng or 2 ng template DNA and 32 cycles of PCR amplification.   The 12 Y-STR systems were investigated in 92 unrelated Japanese males (Hiroshima Prefecture). Gene diversity and haplotype diversity values were calculated from the allelic frequency for each locus. The DYS385 locus was proved to be highly polymorphic (0.9451), and the other loci showed gene diversity values ranging from 0.1229 to 0.7863. A total of 88 haplotypes was observed and the haplotype diversity was calculated to be 0.9990.   These results suggested that the PowerPlex Y System was possible to discriminate most of the male individuals in the Japanese population and can be used in forensic investigation, such as analysis of male/male or male/female DNA mixture and decomposed DNA samples.