In sexual assault cases, the evidence sample, such as fingernail scrapings, may have minimal amounts of foreign DNA from a male assailant. In some cases, the autosomal STR results contain predominantly female alleles mixed with alleles from the male suspect at minimal levels; this makes the interpretation of the results difficult, if not impossible. When male and female biological material is mixed together, polymorphic STR loci that reside on the nonrecombining region of the Y-chromosome may provide useful information. Thus Y-chromosome specific STR loci have become increasingly important in investigating difficult cases. Further, the haploid nature of Y-specific genetic markers may aid in the genetic characterization of male contributors in a multiple-source DNA sample. The Y-PLEXtm5 enables simultaneous amplification of five polymorphic short tandem repeat (STR) loci that reside on the Y-chromosome. These are DYS3891, DYS389II, DYS439, DYS438, and DYS392. As little as 0.1 ng of template DNA can be used for analysis. The specificity of the amplification reaction permitted the analysis of male DNA in a male-female DNA mixture at a ratio of 1:600. Mean stutter values ranged from 3.60 to 10.97 percent. Among the primates investigated, the DNA from an orangutan had amplification at DYS438 locus and from a gorilla at DYS439 and DYS438 loci. The DNA from a cat, a dog, and a horse did not yield any amplified product. Studies on the development of the genotyping system, generation and description of the allelic ladder, and validation of the multiplex PCR according to the FBI Director's Quality Assurance Standards were conducted. Y-STR allele and haplotype frequencies in two populations were generated. The data indicate that the Y-PLEXtm5 genotyping system is sufficiently sensitive and reliable for use in human forensic and male lineage identification cases. 9 tables, 8 figures, and 26 references