Y-chromosome short tandem repeats (Y-STRs) provide valuable information in cases of rape and questioned paternity, and they allow for the genetic identification of male lineages. The present study validated a Y-STR 10-plex on the ABI PRISM 3100 Genetic Analyzer for use in forensic and paternity laboratories at Orchid Cellmark. Following optimization of the polymerase chain reaction, father-son pairs were analyzed to ensure that each pair generated identical haplotypes. This study demonstrated that the 10-plex is sensitive to 0.125 ng of input DNA and that female samples mixed with male samples did not interfere with Y-STR haplotyping. In a sample of 525 males, there were three instances of locus multiplication, two at DYS19 and one at DYS435. Overall, haplotype diversity was 0.996, suggesting that the 10-plex can effectively distinguish among male lineages.