A genotyping system, Y-PLEX 5, has been developed for use in human identification. The Y-PLEX 5 enables simultaneous amplification of five polymorphic short tandem repeat (STR) loci residing on the Y-chromosome, which are DYS389I, 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 enabled analysis of male DNA in a male: female DNA mixture at a ratio of 1:600. Mean stutter values ranged from 3.60-10.97%. Among the different primates investigated, the DNA from orangutan exhibited amplification at DYS438 locus and from gorilla at DYS439 and DYS438 loci. The DNA from cat, dog, and horse did not yield any amplified product. Studies on development of the genotyping system, generation and description of the allelic ladder, and validation of the multiplex PCR as per the FBI Director's Quality Assurance Standards were carried out. Y-STR allele and haplotype frequencies in two populations were generated. The data indicate that the Y-PLEX 5 genotyping system is sensitive and reliable, and can be used in human forensic and male lineage identification cases.